Purification of isoprene from renewable resources

ABSTRACT

Methods and apparatus for the purification of isoprene, such as the purification of a bioisoprene composition from fermentor off-gas. The apparatus includes two columns that process the fermentor off-gas, which includes isoprene and various impurities. A solvent is added to the off-gas in the first column, and the isoprene is stripped from the solvent in the second column. Also provided is a downstream further purification process. Also provided are the resulting purified isoprene compositions.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.61/288,142, filed Dec. 18, 2009, incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

This disclosure relates to production of isoprene.

BACKGROUND

Isoprene (2-methyl-1,3-butadiene) is an important organic compound usedin a wide array of applications. For instance, isoprene is employed asan intermediate or a starting material in the synthesis of numerouschemical compositions and polymers. Isoprene is also an importantbiological material that is synthesized naturally by many plants andanimals, including humans.

Isoprene became an important monomer for utilization in the synthesis ofcis-1,4-polyisoprene when its stereo-regulated polymerization becamecommercially possible in the early 1960s. cis-1,4-Polyisoprene made bysuch stereo-regulated polymerizations is similar in structure andproperties to natural rubber. Even though it is not identical to naturalrubber, it can be used as a substitute for natural rubber in manyapplications. For instance, synthetic cis-1,4-polyisoprene rubber iswidely used in manufacturing vehicle tires and other rubber products.This demand for synthetic cis-1,4-polyisoprene rubber consumes amajority of the isoprene available in the worldwide market. Theremaining isoprene is used in making other synthetic rubbers, blockcopolymers, and other chemical products. For instance, isoprene is usedin making butadiene-isoprene rubbers, styrene-isoprene copolymerrubbers, styrene-isoprene-butadiene rubbers, styrene-isoprene-styreneblock copolymers, and styrene-isoprene block copolymers.

Over the years, many synthesis routes for producing isoprene have beeninvestigated. For instance, the synthesis of isoprene by reactingisobutylene with formaldehyde in the presence of a catalyst is describedin U.S. Pat. Nos. 3,146,278, 3,437,711, 3,621,072, 3,662,016, 3,972,955,4,000,209, 4,014,952, 4,067,923, and 4,511,751. U.S. Pat. No. 3,574,780discloses another process for the manufacture of isoprene by passing amixture of methyl-tert-butyl ether and air over mixed oxide catalysts.The methyl-tert-butyl ether is then cracked into isobutylene andmethanol over the catalyst. The methanol produced is oxidized intoformaldehyde which then reacts with the isobutylene over the samecatalyst to produce the isoprene. U.S. Pat. No. 5,177,290 discloses aprocess for producing dienes, including isoprene, which involvesreacting a reaction mixture of a tertiary alkyl ether and a source ofoxygen over two functionally distinct catalysts under reactionconditions sufficient to produce high yields of the dienes with minimalrecycle of the tertiary alkyl ether and tertiary alkyl etherdecomposition products.

The isoprene used in industrial applications is typically produced as aby-product of the thermal cracking of petroleum or naphtha or isotherwise extracted from petrochemical streams. This is a relativelyexpensive energy-intensive process. With the worldwide demand forpetrochemical based products constantly increasing, the cost of isopreneis expected to rise to much higher levels in the long-term and itsavailability is limited in any case. There is concern that futuresupplies of isoprene from petrochemical-based sources will be inadequateto meet projected needs and that prices will rise to unprecedentedlevels. Accordingly, there is a need to procure a source of isoprenefrom a low cost, renewable source which is environmentally friendly.

Several recent advancements have been made in the production of isoprenefrom renewable sources (see, for example, International PatentApplication Publication No. WO2009/076676). These production techniquesoften results in isoprene compositions containing various amounts ofimpurities as part of the fermentation process. For example,fermentation may generate volatile components, such as water vapor fromthe fermentation media, carbon dioxide as a respiration product, andresidual oxygen in case of aerobic metabolism, as well as other organicbio-byproducts. Oxygen may initiate unwanted chemical reactions ofisoprene, reducing yield and generating undesirable reaction products.Carbon dioxide is a known inhibitor for subsequent catalytic reactionsfor conversion and application of isoprene, such as isoprene topolymers, such as dimers, trimers, up to very long-chained polymers suchas synthetic rubber. Water vapor and other residual bio-byproducts arealso undesirable for many applications using isoprene. Accordingly,purification techniques and methods for isoprene compositions producedfrom renewable resources are desirable.

The disclosures of all publications, patents, patent applications andpublished patent applications referred to herein are hereby incorporatedherein by reference in their entirety.

SUMMARY

The present disclosure provides, inter alia, methods and apparatus forpurifying isoprene from renewable resources or similar and the resultingpurified isoprene compositions.

In one aspect there is provided a method of purifying isoprene from afermentor off-gas, wherein the off-gas comprises isoprene, volatileimpurity, and bio-byproduct impurity, the method comprising: contactingthe fermentor off-gas with a solvent in an apparatus including a firstcolumn to form: an isoprene-rich solution comprising the solvent, amajor portion of the isoprene and a major portion of the bio-byproductimpurity; and a vapor comprising a major portion of the volatileimpurity; transferring the isoprene-rich solution from the first columnto a second column; and stripping isoprene from the isoprene-richsolution in the second column to form: an isoprene-lean solutioncomprising a major portion of the bio-byproduct impurity; and a purifiedisopene composition. In some embodiments, the off-gas is a bioisoprenecomposition.

In any of these embodiments, the volatile impurity comprises a compoundselected from H₂O, CO₂, N₂, H₂, CO and O₂. In some embodiments, thevolatile impurity comprises H₂O, CO₂, and N₂. In some embodiments, thevolatile impurity comprises about 25 to about 80 mol % CO₂, about 45 toabout 99 mol % N₂, and optionally comprises less than about 50 mol % O₂.In some embodiments, the volatile impurity comprises about 40 to about60 mol % CO₂, about 65 to about 99 mol % N₂, and optionally comprisesless than about 25 mol % O₂.

In any of these embodiments, the bio-byproduct impurity comprises apolar or non- or semi-polar impurity. In some embodiments, thebio-byproduct impurity comprises one, two, three, or more compoundsselected from ethanol, acetone, methanol, acetaldehyde, methacrolein,methyl vinyl ketone, 3-methylfuran, 2-methyl-2-vinyloxirane, cis- andtrans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol (such as3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), 2-heptanone,6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine,2,3,5-trimethylpyrazine, citronellal, methanethiol, methyl acetate,1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate,2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol,2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal,butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate,3-methyl-2-buten-1-yl acetate, (E)-3,7-dimethyl-1,3,6-octatriene,(Z)-3,7-dimethyl-1,3,6-octatriene,(E,E)-3,7,11-trimethyl-1,3,6,10-dodecatetraene and(E)-7,11-dimethyl-3-methylene-1,6,10-dodecatriene, 3-hexen-1-ol,3-hexen-1-yl acetate, limonene, geraniol(trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol(3,7-dimethyl-6-octen-1-ol), (E)-3-methyl-1,3-pentadiene,(Z)-3-methyl-1,3-pentadiene. In some embodiments, in the fermentedoff-gas the amount of bio-byproduct relative to amount of isoprene isgreater than about 0.01% (w/w), or greater than about 0.05% (w/w).

In any of these embodiments, the solvent is a non-polar high-boilingpoint solvent. In some embodiments, the solvent has a boiling point ofgreater than about 350° F., or greater than about 375° F. In someembodiments, the solvent has a CO₂ Ostwald coefficient at 130° F. ofless than about 1.25, or less than about 1.1. In some embodiments, thesolvent has a Kauri-butanol value of less than about 50, or from about20 to about 30, or from about 23 to about 27. In some embodiments, thesolvent has an Aniline Point of greater than about 150° F., or fromabout 175° F. to about 200° F. In some embodiments, the solvent has akinematic viscosity at 40° C. is less than about 2.5 centistokes (cSt),or less than about 1.75 centistokes (cSt). In some embodiments, thesolvent has a surface tension at 25° C. from about 20 to 30 dyne/cm, orabout 23 to 27 dyne/cm. In some embodiments, the solvent has an averagemolecular weight from about 125 to about 225 u, or from about 140 toabout 200 u (hereinafter without the “u”). In some embodiments, thesolvent is a selected from a terpene, a paraffin, a monoaromatichydrocarbon, a polyaromatic hydrocarbon, or a mixture thereof. In someembodiments, the solvent is a paraffin (e.g., a C10-C20 paraffin, suchas a C12-C14 paraffin). In some embodiments, the solvent is selectedfrom a solvent substantially similar to Isopar™ L, Isopar™ H and Isopar™M. In some embodiments, the solvent is selected from Isopar™ L, Isopar™H and Isopar™ M. In some embodiments, the solvent is substantiallysimilar to Isopar™ L. In some embodiments, the solvent is Isopar' L. Insome embodiments, the solvent further comprises a polymerizationinhibitor. In some embodiments, the polymerization inhibitor is selectedfrom 2,2,6,6-Tetramethylpiperidine 1-oxyl (TEMPO);4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL);Bis(1-oxyl-2,2,6,6-tetramethylpiperidine-4-yl)sebacate (bridged TEMPO);and t-butyl catechol. In some embodiments, the concentration of thepolymerization inhibitor is from about 0.001% to about 0.1% (w/w)relative to the concentration of isoprene.

In any of these embodiments, the temperature of the fermentor off-gas isreduced prior to contacting the solvent in the first column.

In any of these embodiments, the fermentor off-gas is transferred to anisolation unit capable of stabilizing the off-gas pressure prior tocontacting the fermentor off-gas with the solvent in the first column.

In any of these embodiments, the fermentor off-gas is at least partiallycondensed prior to contacting the solvent in the first column.

In any of these embodiments, the step of contacting the fermentoroff-gas with a solvent in a first column comprises cooling the feedsolvent. The lean solvent stream is cooled or chilled before being fedto the first column, e.g., to 4° C. (40° F.).

In some embodiments, the bottom stream from the first (or second) columnis reboiled to greater than about 66° C. (150° F.), or greater thanabout 91° C. (200° F.). In some embodiments, that bottom stream isreboiled from about 93° C. (200° F.) to about 135° C. (275° F.), or fromabout 110° C. (230° F.) to about 121° C. (250° F.). The reboiling stripsCO₂, which is a volatile impurity, from the isoprene rich solvent.

In any of these embodiments, the step of contacting the fermentoroff-gas with a solvent in a first column further comprises adding steamto the first column as an alternative to reboiling the bottom stream,which is necessary under certain operating conditions.

In any of these embodiments, the step of stripping isoprene from theisoprene-rich solution in the second column comprises adding steam tothe second column as an alternative to the reboiling.

In any of these embodiments, the method further comprises transferringthe purified isoprene-lean solution to the first column for reuse. Insome embodiments, the method further comprises: purifying theisoprene-lean solution to remove a major portion of the bio-byproductimpurity; and transferring the purified isoprene-lean solution to thefirst column for reuse. In some embodiments, purifying the isoprene-leansolution comprises treating the isoprene-lean solution with anadsorption system. In some embodiments, the adsorption system comprisesactivated carbon, alumina, silica, or Selexsorb® (from BASF). In someembodiments, the adsorption system comprises silica. In someembodiments, purifying the isoprene-lean solution comprisesdistillation. In some embodiments, purifying the isoprene-lean solutioncomprises liquid-liquid extraction.

In any of these embodiments, the temperature of the isoprene-leansolution is reduced prior to removing a major portion of thebio-byproduct impurity. In some embodiments, the temperature of theisoprene-lean solution is reduced to less than about 66° C. (150° F.),or to less than about 38° C. (100° F.), or to less than about 24° C.(75° F.).

In any of these embodiments, the method comprises further purifying thepurified isoprene composition. In some embodiments, purifying theisoprene comprises distillation (e.g., after the purified isoprenecomposition is transferred from the second column to a refluxcondenser). In some embodiments, further purifying the purified isoprenecomposition comprises treating the purified isoprene composition with anadsorption system. In some embodiments, the adsorption system comprisesactivated carbon, alumina, silica, or Selexsorb®. In some embodiments,the adsorption system comprises silica.

In any of these embodiments, the method further comprises removing fromvapor a minor portion of the isoprene, if present. In some embodiments,removing a minor portion of the isoprene, if present, comprises treatingvapor with an adsorption system. In some embodiments, the adsorptionsystem comprises activated carbon, alumina, silica, or Selexsorb®. Insome embodiments, the adsorption system comprises activated carbon.

In any of these embodiments, the fermentor off-gas is provided to thefirst column at greater than atmospheric pressure.

In any of these embodiments, the purified isoprene composition has apurity of greater than about 90%, or greater than about 95%, or greaterthan about 99%.

In any of these embodiments, the purified isoprene composition comprisesless than about 25% bio-byproduct impurity relative to the bio-byproductimpurity of the fermentor off-gas, or less than about 10%, or less thanabout 5%.

In any of these embodiments, the purified isoprene composition comprisesless than about 2.5% water and 0.25% CO₂, O₂, and N₂ as volatileimpurities relative to the volatile impurity of the fermentor off-gas,or less than about 0.10%, or less than about 0.05% of these impurities.

In another aspect is provided a purified isoprene composition preparableby any one of the methods described herein. In some embodiments there isprovided purified isoprene composition prepared by any one of themethods described herein.

In another aspect there is provided an isoprene composition. In someembodiments, the composition comprises isoprene, and bio-byproductimpurity, wherein the bio-byproduct impurity comprises C5 hydrocarbons,and there is greater than about 99.94% isoprene (w/w) relative to theweight of C5 hydrocarbons, and less than about 0.05% bio-byproduct (w/w)relative to the weight of the isoprene. In some embodiments, thebio-byproduct comprises one or more compounds as listed above, andincluding those selected from the group consisting of 2-heptanone,6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine,2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methylacetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethylacetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone,1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate,3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate,3-methyl-3-but-1-enyl acetate, 3-methyl-2-but-1-enyl acetate,(E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene,and 2,3-cycloheptenolpyridine. In some embodiments, the compositioncomprises less than about 5% volatile impurity relative to the weight ofthe composition. In some embodiments, the composition comprises isopreneat greater than about 95% relative to the weight of the composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of a process and associated apparatus for purifyingisoprene as described herein.

FIG. 2 is a plot of isoprene absorption efficiency.

FIG. 3 is an analysis of isoprene/solvent composition.

FIG. 4 shows detail of FIG. 3.

FIG. 5 is a plot of isoprene recovered from a solution.

FIG. 6 is a diagram of a process and associated apparatus to furtherpurify isoprene.

FIG. 7 is a plot of impurities in isoprene.

FIG. 8 is a plot of a concentration of the impurity dimethyl disulfideover time.

FIG. 9 is another plot of dimethyl disulfide concentration over time.

DETAILED DESCRIPTION

This disclosure provides, inter alia, methods and apparatus forpurifying isoprene from renewable resources. These methods may use oneor more columns to remove volatile and/or bio-byproduct impuritiesresulting from fermentation.

We have determined methods of purifying isoprene in a fermentor off-gasgenerated from renewable resources using solvents (e.g., non-polarsolvents) with absorption and stripping processes that may provideisoprene having significantly improved purity. The purified isoprenecompositions described herein are particularly suitable for use inapplications conventionally using petroleum-based isoprene, such aspolymerization and use as a starting material in the synthesis ofnumerous desirable chemical compositions.

Accordingly, in one aspect is provided a method of purifying isoprenefrom a fermentor off-gas, comprising: contacting the fermentor off-gaswith a solvent in a column to form: an isoprene-rich solution comprisingthe solvent and a major portion of the isoprene; and a vapor comprisinga major portion of the volatile impurity. In some embodiments, themethod further comprises: stripping isoprene from the isoprene-richsolution in a second column to form: an isoprene-lean solutioncomprising a major portion of the bio-byproduct impurity; and purifiedisoprene composition. Also provided are purified isoprene compositions.

Unless expressed otherwise herein, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention pertains.

As used herein, the singular terms “a,” “an,” and “the” include theplural reference unless the context clearly indicates otherwise.

It is intended that every maximum numerical limitation given throughoutthis specification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

The term “isoprene” refers to 2-methyl-1,3-butadiene (CAS# 78-79-5).Isoprene can be produced as the direct and final volatile C5 hydrocarbonproduct from the elimination of pyrophosphate from 3,3-dimethylallylpyrophosphate (DMAPP), and does not involve the linking orpolymerization of [an] IPP molecule(s) to [a] DMAPP molecule(s). Theterm “isoprene” is not generally intended to be limited to its method ofproduction unless indicated otherwise herein.

As used herein, “biologically produced isoprene” or “bioisoprene” refersto isoprene produced by any biological means, such as produced bygenetically engineered cell cultures, natural microbials, plants oranimals. A bioisoprene composition usually contains fewer hydrocarbonimpurities than isoprene produced from petrochemical sources and oftenrequires minimal treatment in order to be of polymerization grade. Abioisoprene composition also has a different impurity profile from apetrochemically produced isoprene composition.

While isoprene can be obtained by fractionating petroleum, thepurification of this material is expensive and time-consuming. Petroleumcracking of the C5 stream of hydrocarbons produces only about 15%isoprene. Isoprene is also naturally produced by a variety of microbial,plant, and animal species. In particular, two pathways have beenidentified for the biosynthesis of isoprene: the mevalonate (MVA)pathway and the non-mevalonate (DXP) pathway. Genetically engineeredcell cultures in bioreactors have produced isoprene more efficiently, inlarger quantities, in higher purities and/or with unique impurityprofiles, e.g., as described in International Patent ApplicationPublication No. WO2009/076676; U.S. patent application Ser. Nos.12/496,573, 12/560,390, 12/560,317, 12/560,370, 12/560,305, and12/560,366; and U.S. provisional patent application Nos. 61/187,930,61/187,934, and 61/187,959.

Crude bioisoprene compositions are distinguished from isoprene derivedfrom petroleum (herein referred to as “petroisoprene”) compositions inthat bioisoprene compositions are substantially free of anycontaminating unsaturated C5 hydrocarbons that are usually present inpetroisoprene compositions, such as, but not limited to,1,3-cyclopentadiene, trans-1,3-pentadiene, cis-1,3-pentadiene,1,4-pentadiene, 1-pentyne, 2-pentyne, 3-methyl-1-butyne,pent-4-ene-1-yne, trans-pent-3-ene-1-yne, and cis-pent-3-ene-1-yne. Ifany contaminating unsaturated C5 hydrocarbons are present in thebioisoprene starting material composition described herein, they arepresent in lower levels than that in petroisoprene compositions. CrudebioIsoprene may have higher levels of certain C5 hydrocarbons thanhighly purified petroisoprene. Several of these impurities areparticularly problematic given their structural similarity to isopreneand the fact that they can act as polymerization catalyst poisons. Asdetailed below, biologically produced isoprene compositions can besubstantially free of any contaminating unsaturated C5 hydrocarbonswithout undergoing extensive purification.

Further, bioisoprene is distinguished from petroisoprene by carbonfinger-printing. In one aspect, bioisoprene has a higher radioactivecarbon-14 (¹⁴C) content or higher ¹⁴C/¹²C ratio that petroisoprene.Bioisoprene is produced from renewable carbon sources, thus the ¹⁴Ccontent or the ¹⁴C/¹²C ratio in bioisoprene is the same as that in thepresent atmosphere. Petroisoprene, on the other hand, is derived fromfossil fuels deposited thousands to millions of years ago, thus the ¹⁴Ccontent or the ¹⁴C/¹²C ratio is diminished due to radioactive decay. Asdiscussed in greater detail herein, the fuel products derived frombioisoprene has higher ¹⁴C content or ¹⁴C/¹²C ratio than fuel productsderived from petroisoprene. In one embodiment, a fuel product derivedfrom bioisoprene described herein has a ¹⁴C content or ¹⁴C/¹²C ratiosimilar to that in the atmosphere. In another aspect, bioisoprene can beanalytically distinguished from petroisoprene by the stable carbonisotope ratio (¹³C/¹²C), which can be reported as “delta values”represented by the symbol δ¹³C. For examples, for isoprene derived fromextractive distillation of C₅ streams from petroleum refineries, δ¹³C isabout −22‰ to about −24‰. This range is typical for light, unsaturatedhydrocarbons derived from petroleum, and products derived frompetroleum-based isoprene typically contain isoprenic units with the sameδ¹³C. Bioisoprene produced by fermentation of corn-derived glucose(δ¹³C-10.73‰) with minimal amounts of other carbon-containing nutrients(e.g., yeast extract) produces isoprene which can be polymerized intopolyisoprene with δ¹³C-14.66‰ to −14.85‰. Products produced from suchbioisoprene are expected to have δ¹³C values that are less negative thanthose derived from petroleum-based isoprene.

Additionally, bioisoprene compositions are distinguished frompetroisoprene composition in that bioisoprene compositions contain otherbio-byproducts, for example comprising polar impurities, that are notpresent or present in much lower levels in petroisoprene compositions,such as alcohols, aldehydes, ketones and the like. The bio-byproduct mayinclude, but is not limited to, ethanol, acetone, methanol,acetaldehyde, methacrolein, methyl vinyl ketone, 3-methylfuran,2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, a C5prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol),2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine,2,3,5-trimethylpyrazine, citronellal, methanethiol, methyl acetate,1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate,2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol,2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal,butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate,3-methyl-2-buten-1-yl acetate, (E)-3,7-dimethyl-1,3,6-octatriene,(Z)-3,7-dimethyl-1,3,6-octatriene, 2,3-cycloheptenolpyridine,3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol(trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol(3,7-dimethyl-6-octen-1-ol) or a linear isoprene polymer (such as alinear isoprene dimer or a linear isoprene trimer derived from thepolymerization of multiple isoprene units). As described herein,bioisoprene compositions may additionally comprise significant amountsof one or more volatile impurities (e.g., O₂, N₂, H₂O, CO₂) acquiredduring fermentation. Removal of one or more of these compounds (e.g.,polar compounds and/or volatile impurities) from the bioisoprene asdescribed in the methods herein may be desirable.

Unless defined otherwise based on the context in which it is used,“major portion” intends an amount greater than 50% (by weight). Forexample, a major portion of isoprene means more than 50% of the isoprenereferenced. In some embodiments, major portion is greater than 60%, 70%,75%, 80%, 90%, 95%, or 99%, by weight.

As used herein, a “purified isoprene composition” refers to an isoprenecomposition that has been separated from at least a portion of one ormore components that are present in the fermentor off-gas (e.g., aportion of volatile impurity and/or bio-byproduct impurity). A purifiedisoprene composition may exist in any phase or mixture of phases, suchas a complete gas phase (e.g., isoprene gas with one or more additionalcomponents), a complete liquid phase (e.g., a solution comprisingisoprene with 0, 1, 2, or more components), a solid phase, or mixturesthereof. In some embodiments, the purified isoprene composition is atleast about 20%, by weight, free from components other than isoprene. Invarious embodiments, the purified isoprene composition is at least orabout 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 98% or 99%, byweight, pure. Purity can be assayed by any appropriate method, e.g., bycolumn chromatography, HPLC analysis, or GC-MS analysis.

As used herein, “bio-byproduct” or “bio-byproduct impurity” refers toone or more organic compounds, excluding isoprene and methane,associated the biological fermentation processes and obtained togetherwith isoprene in the referenced fermentor off-gas.

As used herein, “volatile impurity” means methane and/or one or moreinorganic compounds found in the referenced fermentor off-gas in thegaseous state under standard atmospheric conditions.

Unless defined otherwise, the meanings of all technical and scientificterms used herein are those commonly understood by one of skill in theart to which this invention belongs. Singleton, et al., Dictionary ofMicrobiology and Molecular Biology, 2nd ed., John Wiley and Sons, NewYork (1994), and Hale & Marham, The Harper Collins Dictionary ofBiology, Harper Perennial, N.Y. (1991) provide one of skill with ageneral dictionary of many of the terms used here. It is to beunderstood that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary. Oneof skill in the art will also appreciate that any methods and materialssimilar or equivalent to those described herein can also be used topractice or test the invention.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to thespecification as a whole.

For use herein, unless clearly indicated otherwise, use of the terms“a”, “an,” and the like refers to one or more.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.” Numeric ranges are inclusive of the numbers defining the range.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

Isoprene Purification

Provided herein are methods of enriching and/or purifying isoprene. Insome embodiments, the isoprene is from a fermentor off-gas. In oneaspect is provided a method of purifying isoprene from a fermentoroff-gas, wherein the off-gas comprises isoprene and volatile impurity.In one embodiment is provided a method of purifying isoprene from afermentor off-gas, wherein the off-gas comprises isoprene and volatileimpurity, the method comprising: contacting the fermentor off-gas with asolvent in a column to form: an isoprene-rich solution comprising thesolvent and a major portion of the isoprene; and a vapor comprising amajor portion of the volatile impurity.

In one aspect is provided a method of purifying isoprene from a solutioncomprising isoprene and bio-byproduct impurity. In one embodiment isprovided a method of purifying isoprene from a solution comprisingisoprene and bio-byproduct impurity, the method comprising: strippingisoprene from the solution in a column to form: an isoprene-leansolution comprising a major portion of the bio-byproduct impurity; and apurified isopene composition.

In one aspect is provided a method of purifying isoprene from afermentor off-gas, wherein the off-gas comprises isoprene, volatileimpurity, and bio-byproduct impurity, the method comprising: (a)contacting the fermentor off-gas with a solvent in a first column toform: an isoprene-rich solution comprising the solvent, a major portionof the isoprene and a major portion of the bio-byproduct impurity; and avapor comprising a major portion of the volatile impurity; (b)transferring the isoprene-rich solution from the first column to asecond column; and (c) stripping isoprene from the isoprene-richsolution in the second column to form: an isoprene-lean solutioncomprising a major portion of the bio-byproduct impurity; and a purifiedisopene composition.

FIG. 1 illustrates an exemplary method of purifying isoprene and anexemplary apparatus. Fermentor off-gas comprising isoprene may begenerated from renewable resources (e.g., carbon sources) by any methodin the art for example, as described in U.S. provisional patentapplication No. 61/187,944, the content of which is hereby incorporatedby reference, particularly with respect to the methods of generatingfermentor off-gas comprising isoprene. The fermentor off-gas generatedfrom one or more individual fermentors 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8,or more fermentors connected in series and/or in parallel) may bedirected to a first column 14. As described below, the fermentor off-gasmay be directed through an isolation unit 16 and/or compressed by acompression means, such as compression system 18. Additionally, thetemperature of the fermentor off-gas may optionally be reduced at anypoint, for example, to form a condensate or partial condensate prior tocontact with the solvent (which may aid in solubilization of one or moreoff-gas components, such as isoprene). The fermentor off-gas may becontacted (e.g., absorbed) at column 14 with a solvent (e.g., anysolvent described herein, such as a non-polar high boiling-pointsolvent). The volatile impurities having less propensity for absorptionin the solvent (particularly with non-polar high boiling-point solvents)are separated from the remaining solvent/fermentor off-gas mixture,resulting in a vapor comprising a major portion of the volatile impurity(e.g., exiting at port 20), and an isoprene-rich solution having a majorportion of the isoprene and a major portion of the bio-byproductimpurity (e.g., at port 22). A stripping vapor flow may be provided byany suitable means (e.g., by steam injection or a reboiler unit 23 belowthe fermentor off-gas feed point in the first column), which may aid inseparation of the volatile impurity from the remaining solution. Steammay be directed through the column (at any suitable location, shown inFIG. 1) to provide a sweeping vapor phase which may aid in the removalof the volatile impurity.

The isoprene-rich solution having a major portion of the isoprene and amajor portion of the bio-byproduct impurity (e.g., at port 22) may bedirected to a second column 24. The second column may be isolated fromthe first column 14 (as shown in FIG. 1) or may be part of a singlecolumn comprising both the first and second columns (e.g., a tandemcolumn wherein the solvent enters the first column at or near one end,and exits the second column at or near an opposite end). The isoprenemay be stripped from the isoprene-rich solution in the second column togenerate a purified isopene composition (e.g., at port 26) and anisoprene-lean solution comprising a major portion of the bio-byproductimpurity (e.g., at port 28). Steam may be added to the second column,which may aid in stripping of the isoprene from the remaining solution.Steam may be directed through the column (at any suitable location, suchas the opposite end of the entry point of the isoprene-rich solutionand/or the near the end of the isoprene-lean solution exit as shown inFIG. 1).

As described herein, the columns may be conventional and of any suitablesize. Exemplary types of columns are commercially available frommanufacturers including Koch Modular Process Systems (Paramus, N.J.),Fluor Corporation (Irving, Tex.), Kuhni USA (Mount Holly, N.C.). Ingeneral, columns are designed to maximize vapor/liquid contact in orderto achieve the desired efficiency. This is achieved by filling thecolumn with either a packing material, or trays spaced at regularintervals along the column. Suitable packing materials include bothrandom and structured types based on metal, glass, polymer and ceramicmaterials. Exemplary random packing types include Raschig rings, Pallrings, A-PAK rings, Saddle rings, Pro-Pak, Heli-Pak, Ceramic saddles andFLEXIRINGS®. Structured packings include wire mesh and perforated metalplate type materials. Manufacturers specializing in column packingsinclude ACS Separations & Mass-Transfer Products (Houston, Tex.),Johnson Bros. Metal Forming Co. (Berkeley, Ill.) and Koch Glitsch, Inc.Knight Div. (East Canton, Ohio). The efficiency of a gas strippingcolumn is expressed in terms of the theoretical plate height and thetotal number of plates in the column. In general, the greater the numberof theoretical plates present, the greater the efficiency of the column.Laboratory scale columns can be purchased from Ace Glass (Vineland,N.J.), Sigma-Aldrich (St. Louis, Mo.) and Chemglass (Vineland, N.J.).Suitable types of glass column include Vigreux, Snyder, Hemple andPerforated-plate type columns. Columns can include packing materials, orcontain features designed to maximize vapor/liquid contact. A laboratoryscale gas scrubber unit (part # CG-1830-10) is available from Chemglassand consists of a packed glass column, solvent reservoir and solventrecirculation pump.

The purified isoprene composition from the second column 24 (e.g.,exiting at port 26) may be further purified by any suitable means (e.g.,by using a reflux condenser 34 and/or an adsorption system 36, such as asilica adsorption system). The reflux reduces the solvent composition inthe isoprene product. The isoprene-lean solution may be recycled back tothe first column for reuse (e.g., as shown in FIG. 1 at port 30). Theisoprene-lean solution may be purified by any suitable means (e.g., byliquid-liquid extraction and/or an adsorption system 32, such as asilica adsorption system) prior to recycling to the first column 14 toreduce to amount of bio-byproduct. Additionally, the temperature of theisoprene-lean solution may be reduced by any suitable means prior torecycling to the first column 14 (e.g., prior to, simultaneously, and/orafter optionally purifying the isoprene solution). FIG. 1 shows anexample of reducing the temperature of the isoprene-lean solution atport 40 prior to purification of the isoprene-lean solution (in thiscase, using coolant for temperature reduction).

In one embodiment, a cooler unit is coupled immediately downstream ofsystem 32 to provide additional cooling. Further, the leanisoprene-solvent from the second column 24 may be phase separated toremove water, before the solution is chilled and returned to the top ofthe first column 14; this phase separation unit would be coupledimmediately below port 40. Further, the condensed water and isoprenefrom condenser 34 may be similarly phase separated to remove water by asimilar phase separator unit coupled immediately downstream of condenser34. Thereby only the isoprene phase is returned back to the secondcolumn. In each case, the water from the phase separation units is awaste stream.

The vapor comprising a major portion of the volatile impurity (e.g., thevapor exiting at port 20 in FIG. 1) may comprise a minor portion ofisoprene (e.g., residual isoprene not remaining in the isoprene-richsolution). The residual isoprene may be recollected for use from thevapor comprising a major portion of the volatile impurity by anysuitable means (e.g., an adsorption system 38, such as an activatedcarbon adsorption system) and in some cases, as shown in FIG. 1, may becombined with the purified isoprene composition (e.g., prior to, during,or after additional purification, such as an adsorption system similarto system 36). FIG. 1 also shows an optional capture device 42 (e.g., athermal oxidizer and/or CO₂ capture system) capable of reducing theamount of undesirable components released into the atmosphere (e.g.,CO₂) from the vapor.

Fermentor Off-Gas

Techniques for producing fermentor off-gas comprising isoprene that maybe used in the methods herein are described in, for example,International Patent Application Publication No. WO2009/076676; U.S.patent application Ser. Nos. 12/496,573, 12/560,390, 12/560,317,12/560,370, 12/560,305, and 12/560,366; and U.S. provisional patentapplication Nos. 61/187,930, 61/187,934, and 61/187,959. In particular,these compositions and methods increase the rate of isoprene productionand increase the amount of isoprene that is produced.

As described in more detail below, the fermentor off-gas may be producedby cells in culture. In some embodiments, the cells in culture arecapable of producing greater than about 400 nmole of isoprene/gram ofcells for the wet weight of the cells/hour (nmole/g_(wcm)/hr) ofisoprene. In some embodiments, the cells have a heterologous nucleicacid that (i) encodes an isoprene synthase polypeptide and (ii) isoperably linked to a promoter. In some embodiments, the cells arecultured in a culture medium that includes a carbon source, such as, butnot limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone,one-carbon source, oil, animal fat, animal oil, fatty acid, lipid,phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride,renewable carbon source, polypeptide (e.g., a microbial or plant proteinor peptide), yeast extract, component from a yeast extract, or anycombination of two or more of the foregoing. In some embodiments, thecells are cultured under limited glucose conditions.

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Exemplary techniques maybe found in Manual of Methods for General Bacteriology Gerhardt et al.,eds), American Society for Microbiology, Washington, D.C. (1994) orBrock in Biotechnology: A Textbook of Industrial Microbiology, SecondEdition (1989) Sinauer Associates, Inc., Sunderland, Mass., which areeach hereby incorporated by reference in their entireties, particularlywith respect to cell culture techniques.

Standard cell culture conditions can be used to culture the cells (see,for example, International Patent Publication WO 2004/033646 andreferences cited therein, which are each hereby incorporated byreference in their entireties, particularly with respect to cell cultureand fermentation conditions). Cells are grown and maintained at anappropriate temperature, gas mixture, and pH (such as at about 20 toabout 37° C., at about 6% to about 84% CO₂, and at a pH between about 5to about 9). In some embodiments, cells are grown at 35° C. in anappropriate cell medium. In some embodiments, e.g., cultures arecultured at approximately 28° C. in appropriate medium in shake culturesor fermentors until desired amount of isoprene production is achieved.In some embodiments, the pH ranges for fermentation are between about pH5.0 to about pH 9.0 (such as about pH 6.0 to about pH 8.0 or about 6.5to about 7.0). Reactions may be performed under aerobic, anoxic, oranaerobic conditions based on the requirements of the host cells.Exemplary culture conditions for a given filamentous fungus are known inthe art and may be found in the scientific literature and/or from thesource of the fungi such as the American Type Culture Collection andFungal Genetics Stock Center.

In various embodiments, the cells are grown using any known mode offermentation, such as batch, fed-batch, or continuous processes. In someembodiments, a batch method of fermentation is used. Classical batchfermentation is a closed system where the composition of the media isset at the beginning of the fermentation and is not subject toartificial alterations during the fermentation. Thus, at the beginningof the fermentation the cell medium is inoculated with the desired hostcells and fermentation is permitted to occur adding nothing to thesystem. Typically, however, “batch” fermentation is batch with respectto the addition of carbon source and attempts are often made atcontrolling factors such as pH and oxygen concentration. In batchsystems, the metabolite and biomass compositions of the system changeconstantly until the time the fermentation is stopped. Within batchcultures, cells moderate through a static lag phase to a high growth logphase and finally to a stationary phase where growth rate is diminishedor halted. In some embodiments, cells in log phase are responsible forthe bulk of the isoprene production. In some embodiments, cells instationary phase produce isoprene.

In some embodiments, the cells in culture are capable of converting morethan about 0.002% of the carbon in a cell culture medium into isoprene.In some embodiments, the cells have a heterologous nucleic acid that (i)encodes an isoprene synthase polypeptide and (ii) is operably linked toa promoter. In some embodiments, the cells are cultured in a culturemedium that includes a carbon source, such as, but not limited to, acarbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source,oil, animal fat, animal oil, fatty acid, lipid, phospholipid,glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbonsource, polypeptide (e.g., a microbial or plant protein or peptide),yeast extract, component from a yeast extract, or any combination of twoor more of the foregoing. In some embodiments, the cells are culturedunder limited glucose conditions.

In some embodiments, the cells in culture comprise a heterologousnucleic acid encoding an isoprene synthase polypeptide. In someembodiments, the cells have a heterologous nucleic acid that (i) encodesan isoprene synthase polypeptide and (ii) is operably linked to apromoter. In some embodiments, the cells are cultured in a culturemedium that includes a carbon source, such as, but not limited to, acarbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source,oil, animal fat, animal oil, fatty acid, lipid, phospholipid,glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbonsource, polypeptide (e.g., a microbial or plant protein or peptide),yeast extract, component from a yeast extract, or any combination of twoor more of the foregoing. In some embodiments, the cells are culturedunder limited glucose conditions.

In some embodiments, the cells in culture are capable of producing anamount of isoprene (such as the total amount of isoprene produced or theamount of isoprene produced per liter of broth per hour per OD₆₀₀)during stationary phase is greater than or about 2 or more times theamount of isoprene produced during the growth phase for the same lengthof time. In some embodiments, the cells in culture are capable ofproducing isoprene only in stationary phase. In some embodiments, thecells in culture are capable of producing isoprene in both the growthphase and stationary phase. In various embodiments, the cells in cultureare capable of producing an amount of isoprene during stationary phaseis greater than or about 2, 3, 4, 5, 10, 20, 30, 40, 50, or more timesthe amount of isoprene produced during the growth phase for the samelength of time.

In some embodiments, the cells in culture are from a system thatincludes a reactor chamber wherein the cells are capable of producinggreater than about 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500,1,750, 2,000, 2,500, 3,000, 4,000, 5,000, or more nmole/g_(wcm)/hrisoprene. In some embodiments, the system is not a closed system. Insome embodiments, at least a portion of the isoprene is removed from thesystem. In some embodiments, the system includes a gas phase comprisingisoprene. In various embodiments, the gas phase comprises any of thecompositions described herein.

In some embodiments, the cells in culture produce isoprene at greaterthan or about 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750,2,000, 2,500, 3,000, 4,000, 5,000, or more nmole/g_(wcm)/hr isoprene. Insome embodiments, the cells in culture convert greater than or about0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6%, or more of the carbon inthe cell culture medium into isoprene. In some embodiments, the cells inculture produce isoprene at greater than or about 1, 10, 25, 50, 100,150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500,1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 100,000, or more ng ofisoprene/gram of cells for the wet weight of the cells/hr(ng/g_(wcm)/h). In some embodiments, the cells in culture produce acumulative titer (total amount) of isoprene at greater than or about 1,10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900,1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000,50,000, 100,000, or more mg of isoprene/L of broth (mg/L_(broth),wherein the volume of broth includes the volume of the cells and thecell medium). Other exemplary rates of isoprene production and totalamounts of isoprene production are disclosed herein.

In some embodiments of any of the aspects, the cells in culture furthercomprise a heterologous nucleic acid encoding an IDI polypeptide. Insome embodiments, the cells further comprise an insertion of a copy ofan endogenous nucleic acid encoding an IDI polypeptide. In someembodiments, the cells further comprise a heterologous nucleic acidencoding a DXS polypeptide. In some embodiments, the cells furthercomprise an insertion of a copy of an endogenous nucleic acid encoding aDXS polypeptide. In some embodiments, the cells further comprise one ormore nucleic acids encoding an IDI polypeptide and a DXS polypeptide. Insome embodiments, one nucleic acid encodes the isoprene synthasepolypeptide, IDI polypeptide, and DXS polypeptide. In some embodiments,one vector encodes the isoprene synthase polypeptide, IDI polypeptide,and DXS polypeptide. In some embodiments, the vector comprises aselective marker, such as an antibiotic resistance nucleic acid.

In some embodiments, the heterologous isoprene synthase nucleic acid isoperably linked to a T7 promoter, such as a T7 promoter contained in amedium or high copy plasmid. In some embodiments, the heterologousisoprene synthase nucleic acid is operably linked to a Trc promoter,such as a Trc promoter contained in a medium or high copy plasmid. Insome embodiments, the heterologous isoprene synthase nucleic acid isoperably linked to a Lac promoter, such as a Lac promoter contained in alow copy plasmid. In some embodiments, the heterologous isoprenesynthase nucleic acid is operably linked to an endogenous promoter, suchas an endogenous alkaline serine protease promoter. In some embodiments,the heterologous isoprene synthase nucleic acid integrates into achromosome of the cells without a selective marker.

In some embodiments, one or more MVA pathway, IDI, DXP, or isoprenesynthase nucleic acids are placed under the control of a promoter orfactor that is more active in stationary phase than in the growth phase.For example, one or more MVA pathway, IDI, DXP, or isoprene synthasenucleic acids may be placed under control of a stationary phase sigmafactor, such as RpoS. In some embodiments, one or more MVA pathway, IDI,DXP, or isoprene synthase nucleic acids are placed under control of apromoter inducible in stationary phase, such as a promoter inducible bya response regulator active in stationary phase.

In some embodiments, at least a portion of the cells in culture maintainthe heterologous isoprene synthase nucleic acid for at least or about 5,10, 20, 40, 50, 60, 65, or more cell divisions in a continuous culture(such as a continuous culture without dilution). In some embodiments,the nucleic acid comprising the isoprene synthase, IDI, or DXS nucleicacid also comprises a selective marker, such as an antibiotic resistancenucleic acid.

In some embodiments, the cells in culture further comprise aheterologous nucleic acid encoding an MVA pathway polypeptide (such asan MVA pathway polypeptide from Saccharomyces cerevisia or Enterococcusfaecalis). In some embodiments, the cells further comprise an insertionof a copy of an endogenous nucleic acid encoding an MVA pathwaypolypeptide (such as an MVA pathway polypeptide from Saccharomycescerevisia or Enterococcus faecalis). In some embodiments, the cellscomprise an isoprene synthase, DXS, and MVA pathway nucleic acid. Insome embodiments, the cells comprise an isoprene synthase nucleic acid,a DXS nucleic acid, an IDI nucleic acid, and a MVA pathway nucleic (inaddition to the IDI nucleic acid).

In some embodiments, the isoprene synthase polypeptide is anaturally-occurring polypeptide from a plant such as Pueraria (e.g.,Pueraria montana or Pueraria lobata).

In some embodiments, the cells in culture are bacterial cells, such asgram-positive bacterial cells (e.g., Bacillus cells such as Bacillussubtilis cells or Streptomyces cells such as Streptomyces lividans,Streptomyces coelicolor, or Streptomyces griseus cells). In someembodiments, the cells in culture are gram-negative bacterial cells(e.g., Escherichia cells such as Escherichia coli cells or Pantoea cellssuch as Pantoea citrea cells). In some embodiments, the cells in cultureare fungal, cells such as filamentous fungal cells (e.g., Trichodermacells such as Trichoderma reesei cells or Aspergillus cells such asAspergillus oryzae and Aspergillus niger) or yeast cells (e.g., Yarrowiacells such as Yarrowia lipolytica cells).

In some embodiments, the microbial polypeptide carbon source includesone or more polypeptides from yeast or bacteria. In some embodiments,the plant polypeptide carbon source includes one or more polypeptidesfrom soy, corn, canola, jatropha, palm, peanut, sunflower, coconut,mustard, rapeseed, cottonseed, palm kernel, olive, safflower, sesame, orlinseed.

As previously mentioned, the fermentor off-gas described herein may bederived from renewable resources (e.g., carbon sources, biologicaland/or plant). Exemplary renewable resources are described in, forexample, U.S. provisional patent application Nos. 61/187,944 (thecontent of which is hereby incorporated by reference), and includecheese whey permeate, cornsteep liquor, sugar beet molasses, barleymalt, and components from any of the foregoing. Exemplary renewablecarbon sources also include acetate, glucose, hexose, pentose and xylosepresent in biomass, such as corn, switchgrass, sugar cane, cell waste offermentation processes, and protein by-product from the milling of soy,corn, or wheat. In some embodiments, the biomass carbon source is alignocellulosic, hemicellulosic, or cellulosic material such as, but arenot limited to, a grass, wheat, wheat straw, bagasse, sugar canebagasse, soft wood pulp, corn, corn cob or husk, corn kernel, fiber fromcorn kernels, corn stover, switch grass, rice hull product, or aby-product from wet or dry milling of grains (e.g., corn, sorghum, rye,triticate, barley, wheat, and/or distillers grains). Exemplarycellulosic materials include wood, paper and pulp waste, herbaceousplants, and fruit pulp. In some embodiments, the carbon source includesany plant part, such as stems, grains, roots, or tubers. In someembodiments, all or part of any of the following plants are used as acarbon source: corn, wheat, rye, sorghum, triticate, rice, millet,barley, cassava, legumes, such as beans and peas, potatoes, sweetpotatoes, bananas, sugarcane, and/or tapioca. In some embodiments, thecarbon source is a biomass hydrolysate, such as a biomass hydrolysatethat includes both xylose and glucose or that includes both sucrose andglucose.

In some embodiments of the methods described herein, the fermentoroff-gas is derived from renewable resources. In some embodiments, thefermentor off-gas comprises bioisoprene. In some embodiments, thefermentor off-gas comprises greater than or about 98.0, 98.5, 99.0,99.5, or 100% isoprene by weight compared to the weight of all C5hydrocarbons in the fermentor off-gas. In some embodiments, thefermentor off-gas comprises greater than or about 99.90, 99.92, 99.94,99.96, 99.98, or 100% isoprene by weight compared to the weight of allC5 hydrocarbons in the fermentor off-gas. In some embodiments, thefermentor off-gas produces a relative detector response of greater thanor about 98.0, 98.5, 99.0, 99.5, or 100% for isoprene compared to thedetector response for all C5 hydrocarbons in the fermentor off-gas whenanalyzed by gas chromatography with flame ionization detection (GC/FID).In some embodiments, the fermentor off-gas produces a relative detectorresponse of greater than or about 99.90, 99.91, 99.92, 99.93, 99.94,99.95, 99.96, 99.97, 99.98, 99.99, or 100% for isoprene compared to thedetector response for all C5 hydrocarbons in the fermentor off-gas whenanalyzed similarly. In some embodiments, the fermentor off-gas comprisesbetween about 98.0 to about 98.5, about 98.5 to about 99.0, about 99.0weight of all C5 hydrocarbons in the fermentor off-gas. In someembodiments, the fermentor off-gas comprises between about 99.90 toabout 99.92, about 99.92 to about 99.94, about 99.94 to about 99.96,about 99.96 to about 99.98, about 99.98 to 100% isoprene by weightcompared to the weight of all C5 hydrocarbons in the fermentor off-gas.

In some embodiments of the methods described herein, the fermentoroff-gas comprises less than or about 2.0, 1.5, 1.0, 0.5, 0.2, 0.12,0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001,0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene,2-methyl-2-butene, 3-methyl-1-butyne, pent-4-ene-1-yne,trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) by weight compared tothe weight of all C5 hydrocarbons in the fermentor off-gas. In someembodiments, the fermentor off-gas has a relative detector response ofless than or about 2.0, 1.5, 1.0, 0.5, 0.2, 0.12, 0.10, 0.08, 0.06,0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% forC5 hydrocarbons other than isoprene compared to the detector responsefor all C5 hydrocarbons in the fermentor off-gas. In some embodiments,the fermentor off-gas has a relative detector response of less than orabout 2.0, 1.5, 1.0, 0.5, 0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01,0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene,3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, orcis-pent-3-ene-1-yne compared to the detector response for all C5hydrocarbons in the fermentor off-gas. In some embodiments, the highlypure isoprene starting composition comprises between about 0.02 to about0.04%, about 0.04 to about 0.06%, about 0.06 to 0.08%, about 0.08 to0.10%, or about 0.10 to about 0.12% C5 hydrocarbons other than isoprene(such 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene,3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, orcis-pent-3-ene-1-yne) by weight compared to the total weight of all C5hydrocarbons in the fermentor off-gas.

In some embodiments of the methods described herein, the fermentoroff-gas comprises less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1,0.05, 0.01, or 0.005 μg/L of a compound that inhibits the polymerizationof isoprene for any compound in the fermentor off-gas that inhibits thepolymerization of isoprene. In some embodiments, the fermentor off-gascomprises between about 0.005 to about 50, such as about 0.01 to about10, about 0.01 to about 5, about 0.01 to about 1, about 0.01 to about0.5, or about 0.01 to about 0.005 μg/L of a compound that inhibits thepolymerization of isoprene for any compound in the fermentor off-gasthat inhibits the polymerization of isoprene. In some embodiments, thefermentor off-gas comprises less than or about 50, 40, 30, 20, 10, 5, 1,0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of a hydrocarbon other than isoprene(such 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene,3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, orcis-pent-3-ene-1-yne). In some embodiments, the fermentor off-gascomprises between about 0.005 to about 50, such as about 0.01 to about10, about 0.01 to about 5, about 0.01 to about 1, about 0.01 to about0.5, or about 0.01 to about 0.005 μg/L of a hydrocarbon other thanisoprene. In some embodiments, the fermentor off-gas comprises less thanor about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/Lof a protein or fatty acid (such as a protein or fatty acid that isnaturally associated with natural rubber).

In some embodiments of the methods described herein, the fermentoroff-gas comprises less than or about 10, 5, 1, 0.8, 0.5, 0.1, 0.05,0.01, or 0.005 ppm of alpha-acetylenes, piperylenes, acetonitrile, or1,3-cyclopentadiene. In some embodiments, the fermentor off-gascomprises less than or about 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm ofsulfur or allenes. In some embodiments, the fermentor off-gas comprisesless than or about 30, 20, 15, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005ppm of all acetylenes (such as 1-pentyne, 2-pentyne, 3-methyl-1-butyne,pent-4-ene-1-yne, trans-pent-3-ene-1-yne, and cis-pent-3-ene-1-yne). Insome embodiments, the fermentor off-gas comprises less than or about2000, 1000, 500, 200, 100, 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05,0.01, or 0.005 ppm of isoprene dimers, such as cyclic isoprene dimers(e.g., cyclic C10 compounds derived from the dimerization of twoisoprene units).

Off-Gas Bio-Byproduct Impurity

The bio-byproduct of the fermentor off-gas may comprise any one or anycombination of compounds described herein. In some embodiments, thebio-byproduct of the fermentor off-gas comprises one or more polarcompounds. Polarity can be determined by methods known in the art, forexample by measuring water solubility, potential for hydrogen bonding,dielectric constant and/or an oil/water partition coefficient. In someembodiments, one or more compounds of the bio-byproduct has an overallpolarity greater than the polarity of isoprene, e.g., as measured byhaving a dielectric constant greater than 2.1 at 25° C. (77° F.). Insome embodiments, greater than about any of 20%, 30%, 50%, 60%, 70%,80%, 90%, or 95% (w/w) of the bio-byproduct is comprised of one or morecompounds having an overall polarity greater than the polarity ofisoprene. In some embodiments, one or more of the compounds of thebio-byproduct has a dielectric constant of greater than about 2, orgreater than about 3, or greater than about 5, or greater than about7.5, or greater than about 10 at 20° C. In some embodiments, greaterthan about any of 20%, 30%, 50%, 60%, 70%, 80%, 90%, or 95% (w/w) of thebio-byproduct is comprised of one or more compounds having a dielectricconstant of greater than about 2, or greater than about 3, or greaterthan about 5, or greater than about 7.5, or greater than about 10 at 20°C.

In some embodiments, the fermentor off-gas includes one or more of thefollowing compounds in the bio-byproduct: an alcohol, an aldehyde, or aketone (such as any of the alcohols, aldehydes, or ketones describedherein). In some embodiments, the fermentor off-gas includes (i) analcohol and an aldehyde, (ii) an alcohol and a ketone, (iii) an aldehydeand a ketone, (iv) an alcohol, an aldehyde, and a ketone, or (v) esters.

The fermentor off-gas may comprise any one or any combination of one ofmore of the following compounds in the bio-byproduct: ethanol, acetone,methanol, acetaldehyde, methacrolein, methyl vinyl ketone,2-methyl-2-vinyloxirane, 3-methylfuran, cis- andtrans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol (such as3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), 2-heptanone,6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine,2,3,5-trimethylpyrazine, citronellal, methanethiol, methyl acetate,1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate,2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol,2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal,butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate,3-methyl-2-buten-1-yl acetate, (E)-3,7-dimethyl-1,3,6-octatriene,(Z)-3,7-dimethyl-1,3,6-octatriene, 2,3-cycloheptenolpyridine,3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol(trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol(3,7-dimethyl-6-octen-1-ol).

In some embodiments, the fermentor off-gas includes any one or anycombination of one of more of the following compounds in thebio-byproduct: 2-heptanone, 6-methyl-5-hepten-2-one,2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal,acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl,2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol,3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone,3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butylacetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate,3-methyl-2-buten-1-yl acetate, (E)-3,7-dimethyl-1,3,6-octatriene,(Z)-3,7-dimethyl-1,3,6-octatriene, 2,3-cycloheptenolpyridine, or alinear isoprene polymer (such as a linear isoprene dimer or a linearisoprene trimer derived from the polymerization of multiple isopreneunits). In some embodiments, the fermentor off-gas comprises one or moreof the following compounds in the bio-byproduct: ethanol, acetone,methanol, acetaldehyde, methacrolein, methyl vinyl ketone,3-methylfuran, 2-methyl-2-vinyloxirane, cis- andtrans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol (such as3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol).

In some embodiments of the methods described herein, the fermentoroff-gas comprises greater than or about 0.005, 0.01, 0.05, 0.1, 0.5, 1,5, 10, 20, 30, 40, 60, 80, 100, or 120 μg/L of bio-byproduct (e.g.,bio-byproduct comprising one or more compounds selected from ethanol,acetone, methanol, acetaldehyde, methacrolein, methyl vinyl ketone,3-methylfuran, 2-methyl-2-vinyloxirane, cis- andtrans-3-methyl-1,3-pentadiene, and a C5 prenyl alcohol (such as3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol)). In some embodiments,the fermentor off-gas comprises greater than or about 0.005, 0.01, 0.05,0.1, 0.5, 1, 5, 10, 20, 30, 40, 60, 80, 100, or 120 μg/L of one or morecompounds in the bio-byproduct (e.g., one or more compounds selectedfrom ethanol, acetone, methanol, acetaldehyde, methacrolein, methylvinyl ketone, 3-methylfuran, 2-methyl-2-vinyloxirane, cis- andtrans-3-methyl-1,3-pentadiene, and a C5 prenyl alcohol (such as3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol)). In some embodiments,the fermentor off-gas comprises between about 0.005 to about 120, suchas about 0.01 to about 80, about 0.01 to about 60, about 0.01 to about40, about 0.01 to about 30, about 0.01 to about 20, about 0.01 to about10, about 0.1 to about 80, about 0.1 to about 60, about 0.1 to about 40,about 5 to about 80, about 5 to about 60, or about 5 to about 40 μg/L ofbio-byproduct (e.g., bio-byproduct comprising one or more compoundsselected from ethanol, acetone, methanol, acetaldehyde, methacrolein,methyl vinyl ketone, 3-methylfuran, 2-methyl-2-vinyloxirane, cis- andtrans-3-methyl-1,3-pentadiene, and a C5 prenyl alcohol (such as3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol)). In some embodiments,the fermentor off-gas comprises between about 0.005 to about 120, suchas about 0.01 to about 80, about 0.01 to about 60, about 0.01 to about40, about 0.01 to about 30, about 0.01 to about 20, about 0.01 to about10, about 0.1 to about 80, about 0.1 to about 60, about 0.1 to about 40,about 5 to about 80, about 5 to about 60, or about 5 to about 40 μg/L ofone or more compounds of the bio-byproduct (e.g., one or more compoundsselected from ethanol, acetone, methanol, acetaldehyde, methacrolein,methyl vinyl ketone, 3-methylfuran, 2-methyl-2-vinyloxirane, cis- andtrans-3-methyl-1,3-pentadiene, and a C5 prenyl alcohol (such as3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol)).

In various embodiments of the methods described herein, the amount ofbio-byproduct and/or the amount of one or more compounds of thebio-byproduct relative to amount of isoprene in units of percentage byweight (i.e., weight of the bio-byproduct divided by the weight ofisoprene times 100) is greater than or about 0.01, 0.02, 0.05, 0.1, 0.5,1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110% (w/w). In someembodiments, the relative detector response for the bio-byproduct and/orone or more compounds of the bio-byproduct compared to the detectorresponse for isoprene is greater than or about 0.01, 0.02, 0.05, 0.1,0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110%. In variousembodiments, the amount of bio-byproduct and/or the amount of one ormore compounds of the bio-byproduct relative to amount of isoprene inunits of percentage by weight (i.e., weight of the bio-byproduct orweight of the one or more compounds divided by the weight of isoprenetimes 100) is between about 0.01 to about 105% (w/w), such as about 0.01to about 90, about 0.01 to about 80, about 0.01 to about 50, about 0.01to about 20, about 0.01 to about 10, about 0.02 to about 50, about 0.05to about 50, about 0.1 to about 50, or 0.1 to about 20% (w/w).

In some embodiments, the fermentor off-gas contains one or more of thefollowing compounds in the bio-byproduct: methanol, ethanol,methanethiol, 1-butanol, 3-methyl-1-propanol, acetone, acetic acid,2-butanone, 2-methyl-1-butanol, or indole. In some embodiments, thefermentor off-gas contains 1 ppm or more of one or more of the followingcompounds: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol,3-methyl-1-propanol, acetone, acetic acid, 2-butanone,2-methyl-1-butanol, or indole. In some embodiments, the concentration ofbio-byproduct and/or one or more compounds of the bio-byproduct (e.g.,of one or more of the following: methanol, acetaldehyde, ethanol,methanethiol, 1-butanol, 3-methyl-1-propanol, acetone, acetic acid,2-butanone, 2-methyl-1-butanol, or indole) is between about 1 to about10,000 ppm in the fermentor off-gas. In some embodiments, the fermentoroff-gas includes one or more of the following: methanol, acetaldehyde,ethanol, methanethiol, 1-butanol, 3-methyl-1-propanol, acetone, aceticacid, 2-butanone, 2-methyl-1-butanol, or indole, at a concentrationbetween about 1 to about 100 ppm, such as about 1 to about 10 ppm, about10 to about 20 ppm, about 20 to about 30 ppm, about 30 to about 40 ppm,about 40 to about 50 ppm, about 50 to about 60 ppm, about 60 to about 70ppm, about 70 to about 80 ppm, about 80 to about 90 ppm, or about 90 toabout 100 ppm. In some embodiments, the amount of bio-byproduct in thefermentor off-gas is at a concentration from between about 1 to about100 ppm, such as about 1 to about 10 ppm, about 10 to about 20 ppm,about 20 to about 30 ppm, about 30 to about 40 ppm, about 40 to about 50ppm, about 50 to about 60 ppm, about 60 to about 70 ppm, about 70 toabout 80 ppm, about 80 to about 90 ppm, or about 90 to about 100 ppm.Bio-byproduct from cell cultures (such as volatile organic compounds inthe headspace of cell cultures) can be analyzed using standard methodssuch as those described herein or other standard methods such as protontransfer reaction-mass spectrometry (see, for example, Bunge et al.,Applied and Environmental Microbiology, 74(7):2179-2186, 2008 which ishereby incorporated by reference in its entirety, particular withrespect to the analysis of volatile organic compounds).

Off-Gas Volatile Impurity

The optimal ranges of various components during the fermentation ofisoprene to achieve suitable production levels and safe operation (e.g.,based on flammability characteristics) is described in, for example,U.S. provisional patent application No. 61/187,944, the content of whichis hereby incorporated by reference. As a result, the fermentationoff-gas may contain volatile impurity (e.g., volatile impuritycomprising water vapor, CO₂, N₂, and O₂). Removing this volatileimpurity from isoprene may be desirable prior to commercial use.Accordingly, in one aspect, the methods described herein decrease orremove volatile impurity from isoprene fermentor off-gas.

In some embodiments, the volatile impurity from fermentor off-gasincludes one, two, or more compounds selected from H₂O, CO₂, CO, N₂,CH₄, H₂, and O₂. In some embodiments, the volatile impurity comprisesH₂O, CO₂, and N₂. In some embodiments, the volatile impurity comprisesH₂O, CO₂, N₂, and O₂. In some embodiments, the volatile impuritycomprises an inorganic gas at standard temperature and pressure.

In some embodiments, the fermentor off-gas comprises volatile impurity(e.g., wherein the volatile impurity comprises a compound such as H₂O,CO₂, CO, N₂, CH₄, H₂, and/or O₂) at a level of at least about 2, 5, 10,50, 75, or 100-fold less than the amount of isoprene. In someembodiments, the volatile impurity of the fermentor off-gas comprisesone or more compounds (e.g., H₂O, CO₂, CO, N₂, CH₄, H₂, and/or O₂) at alevel of at least about 2, 5, 10, 50, 75, or 100-fold less than theamount of isoprene. In some embodiments, the portion off-gas other thanisoprene comprises between about 0% to about 100% (volume) oxygen, suchas between about 0% to about 10%, about 10% to about 20%, about 20% toabout 30%, about 30% to about 40%, about 40% to about 50%, about 50% toabout 60%, about 60% to about 70%, about 70% to about 80%, about 80% toabout 90%, or about 90% to about 100% (volume) oxygen. In someembodiments, the portion of off-gas other than isoprene comprisesbetween about 0% to about 99% (volume) nitrogen, such as between about0% to about 10%, about 10% to about 20%, about 20% to about 30%, about30% to about 40%, about 40% to about 50%, about 50% to about 60%, about60% to about 70%, about 70% to about 80%, about 80% to about 90%, orabout 90% to about 99% (volume) nitrogen. In some embodiments, theportion of off-gas other than isoprene comprises between about 0% toabout 99% (volume) H₂O, such as between about 0% to about 10%, about 10%to about 20%, about 20% to about 30%, about 30% to about 40%, about 40%to about 50%, about 50% to about 60%, about 60% to about 70%, about 70%to about 80%, about 80% to about 90%, or about 90% to about 99% (volume)H₂O. In some embodiments, the portion off-gas other than isoprenecomprises between about 1% to about 50% (volume) CO₂, such as betweenabout 1% to about 10%, about 10% to about 20%, about 20% to about 30%,about 30% to about 40%, or about 40% to about 50% (volume) CO₂.

In some embodiments, the volatile impurity of the fermentor off-gascomprises about 10 to about 90, or about 20 to about 80, or about 40 toabout 60, or about 10 to about 20, or about 20 to about 30, or about 30to about 40, or about 40 to about 50, or about 50 to about 60, or about60 to about 70, or about 70 to about 80, or about 80 to 90, or about 90to about 99 mol % N₂. In some embodiments, the volatile impuritycomprises about 10 to about 90, or about 20 to about 80, or about 40 toabout 60, or about 10 to about 20, or about 20 to about 30, or about 30to about 40, or about 40 to about 50, or about 50 to about 60, or about60 to about 70, or about 70 to about 80 or about 90, or about 90 toabout 99 mol % carbon dioxide. In some embodiments, the volatileimpurity comprises about 10 to about 90, or about 20 to about 80, orabout 40 to about 60, or about 10 to about 20, or about 20 to about 30,or about 30 to about 40, or about 40 to about 50, or about 50 to about60, or about 60 to about 70, or about 70 to about 80 or about 90, orabout 90 to about 99 mol % carbon monoxide. In some embodiments, thevolatile impurity comprises about 10 to about 90, or about 20 to about80, or about 40 to about 60, or about 10 to about 20, or about 20 toabout 30, or about 30 to about 40, or about 40 to about 50, or about 50to about 60, or about 60 to about 70, or about 70 to about 80 or about90, or about 90 to about 99, or less than 50, or less than 40, or lessthan 30, or less than 20, or less than 10, or less than 5, or zero, orgreater than 80, or greater than 90, or greater than 95 mol % O₂. Insome embodiments, the volatile impurity comprises about 10 to about 90,or about 20 to about 80, or about 40 to about 60, or about 10 to about20, or about 20 to about 30, or about 30 to about 40, or about 40 toabout 50, or about 50 to about 60, or about 60 to about 70, or about 70to about 80 or about 90, or about 90 to about 99 mol % hydrogen. In someembodiments, the volatile impurity comprises less than about 50, or lessthan about 40, or less than about 30, or less than about 20, or lessthan about 10, or less than about 5, or less than about 3 mol % methane.

In some embodiments, the volatile impurity of the fermentor off-gascomprises about 25 to about 80 mol % CO₂, about 45 to about 99 mol % N₂,and optionally comprises less than about 50 mol % O₂. In someembodiments, the volatile impurity comprises about 40 to about 60 mol %CO₂, about 65 to about 99 mol % N₂, and optionally comprises less thanabout 25 mol % O₂.

Although the fermentor off-gas derived from renewable resourcesoriginates from fermentation in the gas phase, the off-gas may exist asdescribed herein in any phase or mixture of phases, such as a completegas phase, in a partial gas phase and partial liquid phase (such as acondensate), or in a complete liquid phase. In some embodiments, atleast a portion of the fermentor off-gas derived from renewableresources is in a gas phase. In some embodiments, at least a portion ofthe fermentor off-gas derived from renewable resources is in a liquidphase (such as a condensate). In some embodiments, at least a portion ofthe fermentor off-gas derived from renewable resources is in a solidphase. In some embodiments, the fermentor off-gas has undergone one ormore purification steps prior to use in the methods described herein. Insome embodiments, the fermentor off-gas has not undergone purificationprior to use in the methods described herein. In some embodiments, atleast a portion of the fermentor off-gas derived from renewableresources is absorbed to a solid support, such as a support thatincludes silica and/or activated carbon prior to use in the methodsdescribed herein. In some embodiments, the fermentor off-gas is mixedwith one or more solvents prior to use in the methods described herein.In some embodiments, the fermentor off-gas is mixed with one or moregases prior to use in the methods described herein.

In some embodiments of the methods described herein, the temperature ofthe fermentor off-gas is reduced prior to contacting the solvent in thefirst column. Temperature reduction of the fermentor off-gas may aid insolubilization of one or more off-gas components (such as isoprene) inthe solvent (e.g., a high boiling point hydrophobic solvent). Thetemperature may be reduced by any suitable means (e.g., use of acoolant). In some embodiments, the temperature reduction of thefermentor off-gas results in a partial or complete condensation of thefermentor off-gas. In some embodiments, the temperature of the fermentoroff-gas is reduced to less than any of about 95%, 90%, 80%, 70%, 60%,50%, 40%, 30%, 20%, or 10% of the off-gas temperature in ° C. from thefermentor(s). In some embodiments, the temperature of the fermentoroff-gas is reduced to less than any of about 150° C., 125° C., 100° C.,90° C., 80° C., 70° C., 60° C., 50° C., 45° C., 40° C., 35° C., 30° C.,25° C., 20° C., 15° C., 10° C., 5° C., or 0° C. In some embodiments, thetemperature of the fermentor off-gas is reduced to any of about 0° C. toabout 150° C., about 0° C. to about 125° C., about 0° C. to about 100°C., about 0° C. to about 75° C., about 0° C. to about 30° C., about 0°C. to about 20° C., about 0° C. to about 10° C., about 0° C. to about7.5° C., or about 5° C.

In some embodiments of the methods described herein, the pressure of thefermentor off-gas is increased prior to contacting the solvent in thefirst column. The pressure may be increased by any suitable means (e.g.,compression systems known in the art). Increased pressure may aid insolubilization of one or more off-gas components (such as isoprene) inthe solvent (e.g., a high boiling point hydrophobic solvent). In someembodiments, the pressure of the fermentor off-gas is increased by morethan any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% ofthe off-gas pressure (in PSIA—pounds per square inch absolute) from thefermentor(s). In some embodiments, the pressure of the fermentor off-gasis increased to more than any of about 10 PSIA, 20 PSIA, 30 PSIA, 40PSIA, 50 PSIA, 60 PSIA, 70 PSIA, 80 PSIA, 90 PSIA, 100 PSIA, 110 PSIA,120 PSIA, 130 PSIA, 140 PSIA, or 150 PSIA. In some embodiments, thepressure of the fermentor off-gas is increased to any of about 5 PSIA toabout 150 PSIA, about 10 PSIA to about 100 PSIA, about 15 PSIA to about75 PSIA, about 20 PSIA to about 65 PSIA, about 25 PSIA to about 60 PSIA,about 30 PSIA to about 50 PSIA, or about 35 PSIA to about 45 PSIA.

Isolation Unit

The fermentor off-gas may be routed through an isolation unit prior toreaching a column. The isolation unit may serve to prevent thefermentation process from being influenced by the downstreampurification process. Additionally, the isolation unit may serve toprovide a stable intermediate pressure to the column (e.g., with amake-up flow of recycle gas stream, fresh atmospheric air and/or otheradded gas, such as nitrogen). Foam-out and entrained liquid (e.g.,media) may also be collected by the isolation unit and prevented fromreaching the column. In some embodiments of any of the methods describedherein, the fermentor off-gas is transferred to an isolation unit (thesame or different isolation unit) prior to contacting the fermentoroff-gas with the solvent in the first column. In some of theseembodiments, the isolation unit is capable of stabilizing the off-gaspressure.

Solvents

Any suitable solvent may be used in the methods described herein. Thesolvent may be a pure solvent or a mixture of two or more solvents. Insome instances, the solvent is capable of absorbing a major portion ofthe isoprene from the fermentor off-gas (or a major portion of theisoprene and a major portion of the bio-byproduct) and is not capable ofabsorbing a major portion of the volatile impurity of the fermentoroff-gas under the same conditions. In some embodiments of the methodsdescribed herein, the solvent is capable of absorbing greater than about2, 5, 10, 20, 50, 100, 200 or 500 times more isoprene (w/w) compared tothe volatile impurity under the same conditions. In some embodiments ofthe methods described herein, the solvent is capable of only arelatively low CO₂ absorption (e.g., as defined by its OstwaldCoefficient). Accordingly, in some embodiments, the solvent is a lowcarbon dioxide absorption solvent. As used herein, unless otherwisestated, a “low carbon dioxide absorption solvent” intends a solventhaving an Ostwald Coefficient of less than 2 at 130° F. and standardpressure. In some embodiments, the solvent is a low carbon dioxideabsorption solvent having a CO₂ Ostwald coefficient of less than aboutany of about 1.75, about 1.5, about 1.25, about 1.1, or about 1.0 at 54°C. (130° F.) and at standard pressure.

The solvent may have a relatively high-boiling point. As used herein,unless otherwise stated, a “high boiling point solvent” intends asolvent having a boiling point of greater than 121° C. (250° F.) at 1atm. In some embodiments of the methods described herein, the solvent isa high boiling point solvent with a boiling point of greater than about121° C. (250° F.), greater than about 135° C. (275° F.), greater thanabout 149° C. (300° F.), greater than about 163° C. (325° F.), greaterthan about 121° C. (350° F.), greater than about 191° C. (375° F.), orgreater than about 204° C. (400° F.), or greater than about 177° C.(420° F.), or greater than about 232° C. (450° F.), or greater thanabout 246° C. (475° F.) at 1 atm. In some embodiments, the solvent has aboiling point from about 121° C. (250° F.) to about 149° C. (300° F.),or 135° C. (275° F.) to about 163° C. (325° F.), or about 149° C. (300°F.) to about 177° C. (350° F.), or about 163° C. (325° F.) to about 149°C. (375° F.), or about 135° C. (350° F.) to about 204° C. (400° F.), orabout (191° C. (375° F.) to about (218° C. (425° F.), or about 204° C.(400° F.) to about 232° C. (450° F.), or about 218° C. (425° F.) toabout 246° C. (475° F.) at 1 atm.

In some embodiments of the methods described herein, the solvent isrelatively non-polar. The polarity of the solvent can be determined byany method known in the art (e.g., water solubility, potential forhydrogen bonding, dielectric constant and/or an oil/water partitioncoefficient). In some embodiments, the solvent is a non-polar solvent.As used herein, unless otherwise stated, a “non-polar solvent” intends asolvent having a dielectric constant of less than 15 at 20° C. In someembodiments, the solvent is a non-polar solvent having a dielectricconstant of less than about 12, or less than about 10, or less thanabout 7.5, or less than about 5, or less than about 3, or less thanabout 2, or less than about 1 at 20° C. In some embodiments, the solventhas a solubility in water of less than about 5%, or less than about 3%,or less than about 2%, or less than about 1%, or less than about 0.5%,or less than about 0.25%, or less than about 0.1%, or less than about0.05%, or less than about 0.025% under standard conditions.

The solvent used in the methods described herein, may be characterizedby its Kauri-butanol value (“Kb value”) as measured in the art. In someembodiments of the methods described herein, the solvent has a Kb valueof less than 75, or less than 50, or less than 40, or less than 30, orless than 20, or less than 10. In some embodiments, the solvent has a Kbvalue from about 10 to about 40, or about 15 to about 35, or about 20 toabout 30, or from about 23 to about 27, or about 25.

The solvent used in the methods described herein, may be characterizedby its Aniline Point as measured in the art. In some embodiments of themethods described herein, the solvent has an Aniline Point of greaterthan about 52° C. (125° F.), or greater than about 66° C. (150° F.), orgreater than about 79° C. (175° F.), or greater than about 91° C. (200°F.). In some embodiments, the solvent has an Aniline Point from about66° C. (150° F.) to about 121° C. (250° F.), or from about 79° C. (175°F.) to about 93° C. (200° F.), or from about 82° C. (180° F.) to about91° C. (195° F.).

The solvent used in the methods described herein, may be characterizedby its Kinematic viscosity as measured in the art. In some embodimentsof the methods described herein, the solvent has a Kinematic viscosityat 40° C. or less than about 3, or less than about 2.75, or less thanabout 2.25, or less than about 2.0, or less than about 1.75, or lessthan about 1.5, or less than about 1.25 centistokes (cSt).

The solvent used in the methods described herein, may be characterizedby its surface tension as measured in the art. In some embodiments ofthe methods described herein, the solvent has a surface tension at 25°C. from about 15 to about 35 dyne/cm, or about 17 to about 32 dyne/cm,or about 20 to about 30 dyne/cm, or about 23 to about 27 dyne/cm, orabout 25 dyne/cm.

The solvent used in the methods described herein, may be characterizedby its molecular weight (or a weighted average molecular weight in thecase of a mixed solvent system). In some embodiments of the methodsdescribed herein, the solvent has an average molecular weight from about100 to about 250, or about 125 to about 225, or about 140 to about 200,or about 150 to about 175.

The solvent used in the methods described herein may have any one orcombination of two or more of the properties described herein. Forexample, in some embodiments, the solvent used in the methods describedherein may be a non-polar, high-boiling point solvent (i.e. a non-polarsolvent that is also a high-boiling point solvent). In some embodiments,the solvent used in the methods described herein may be a non-polar,low-carbon dioxide absorption solvent; or a low-carbon dioxideabsorption, high-boiling point solvent; or a non-polar, low-carbondioxide absorption, high-boiling point solvent. In some embodiments ofthe methods described herein, the solvent is characterized as having aboiling point of greater than about 177° C. (350° F.), a solubility inwater of less than about 3%, and a CO₂ Ostwald coefficient of less thanabout 1.25 at 54° C. (130° F.). In some of these embodiments, thesolvent has an average molecular weight from about 125 to about 225. Inother embodiments of the methods described herein, the solvent ischaracterized as having a boiling point of greater than about 191 C(375° F.), a solubility in water of less than about 1%, and a CO₂Ostwald coefficient of less than about 1.1 at 54° C. (130° F.). In someof these embodiments, the solvent has an average molecular weight fromabout 140 to about 200.

In some embodiments of the methods described herein, the solvent is aselected from a terpene, a paraffin, a monoaromatic hydrocarbon, apolyaromatic hydrocarbon, or a mixture thereof. In some embodiments, thesolvent is a paraffin (e.g., a C10-C20 paraffin, such as a C12-C14paraffin) or an isoparaffin as described above. In some embodiments, thesolvent is a terpene. In some embodiments, the solvent is a monoaromatichydrocarbon. In some embodiments, the solvent is a polyaromatichydrocarbon. In some embodiments, the solvent is an Isopar™ solvent(commercially available from Exxon) or equivalent thereof, such as asolvent substantially similar to any solvent described in Table 1 (e.g.,solvent 1, 2, 3, 4, 5, 6, 7, and/or 8). In some embodiments, the solventhas any one or more properties substantially similar to any solventdescribed in Table 1 (e.g., solvent 1, 2, 3, 4, 5, 6, 7, and/or 8). Insome embodiments, the solvent is selected from Isopar™ L (Table 1;solvent 6), Isopar™ H (Table 1; solvent 4) and Isopar™ M (Table 1;solvent 7). In some embodiments, the solvent is Isopar™ L (Table 1;solvent 6). In some embodiments, the solvent is Isopar™ H (Table 1;solvent 4). In some embodiments, the solvent is Isopar™ M (Table 1;solvent 7).

TABLE 1 Solvent 1 2 3 4 5 6 7 8 Tradename Isopar ™ C Isopar ™ E Isopar ™G Isopar ™ H Isopar ™ K Isopar ™ L Isopar ™ M Isopar ™ V Kauri-butanol27 29 27 26 27 27 25 23 value¹ Aniline Point 173 167 181 183 181 185 196198 (° F.) Flash Point 18 45 106 129 135 147 199 265 (° F.)²Distillation 208 244 320 352 351 372 433 523 (° F.)³ Distillation 219279 349 370 387 405 489 594 (° F.)⁴ Specific 0.70 0.72 0.75 0.76 0.760.77 0.79 0.83 Gravity (@60° F.)⁵ Saturates 100 100 100 100 99.9 99.999.9 99.8 Aromatics <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.05 <0.5 Acids(ppm) None None None None None None None None Chlorides <3 <2 <1 <3 2 <1— 7 (ppm) Nitrogen — <2 <1 <1 <1 <1 — — (ppm) Peroxides 0 0 Trace <1 <1<1 <1 <1 (ppm) Sulfur 1 1 1 1 <2 <2 <2 1 (ppm) Surface 20.3 22.1 23.824.1 24.2 25.1 26.4 26.9 tension (@77° F.; dynes/cm)⁶ Interfacial 48.948.9 51.6 51.4 50.1 49.8 52.2 44.9 tension (@77° F.) DemulsibilityExcellent Excellent Excellent Excellent Excellent Excellent ExcellentExcellent ¹ASTM D1133; ²ASTM D56, TTC; ³ASTM D86, IBP; ⁴ASTM D86, DryPoint; ⁵ASDM D1250; ⁶ASTM D971

The solvent used in the methods described herein may further comprise apolymerization inhibitor to aid in reducing unwanted polymerization ofisoprene. Accordingly, in some embodiments, the solvent furthercomprises a polymerization inhibitor (e.g., an inhibitor of isoprenepolymerization). Suitable inhibitors include, for example,2,2,6,6-Tetramethylpiperidine 1-oxyl (TEMPO);4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL);Bis(1-oxyl-2,2,6,6-tetramethylpiperidine-4-yl)sebacate (bridged TEMPO);and t-butyl catechol. In some embodiments of the methods describedherein, the solvent comprises a suitable amount of a polymerizationinhibitor to sufficiently prevent polymerization of isoprene (e.g., toprevent more than 95%, or more than 97%, or more than 98%, or more than99%, or more than 99.5% of the isoprene from polymerizing compared toabsence of the inhibitor). In some embodiments, the solvent comprises apolymerization inhibitor at a concentration from about 0.001% to about0.1%, or from about 0.005% to about 0.075%, or from about 0.01% to about0.05% (w/w) relative to isoprene. In some embodiments, the solventcomprises a polymerization inhibitor at a concentration from about0.001% to about 0.1%, or from about 0.005% to about 0.075%, or fromabout 0.01% to about 0.05% (w/w) relative to solvent.

Removal of Volatile Gases

In some aspects, the methods described herein include removing volatilegases from a fermentor off-gas. In some of the embodiments describedherein, the fermentor off-gas is contacted with a solvent in a column.In some of these embodiments, the fermentor off-gas is contacted with asolvent in a column to form: an isoprene-rich solution comprising thesolvent and a major portion of the isoprene; and a vapor comprising amajor portion of the volatile impurity. A stripping vapor flow may beintroduced in to the column (e.g., the first column) below the fermentoroff-gas feed point, which may aid in separation of the volatile impurityfrom the remaining solution. The stripping vapor may be introduced byany suitable means (e.g., steam or the column bottom reboiler). In someembodiments, the temperature of the column bottom stream (e.g., at thefirst column) is much greater than the temperature of the solvent priorto entering the column. In some embodiments, the temperature of thecolumn bottom stream (e.g., at the first column) is greater by any ofabout 38° C. (100° F.), 52° C. (125° F.), 66° C. (150° F.), 79° C. (175°F.), 93° C. (200° F.), 109° C. (225° F.), 121° C. (250° F.), 135° C.(275° F.), or 149° C. (300° F.). In some embodiments, the temperature ofthe solvent in the column bottom stream (e.g., at the first column) isfrom about 66° C. (150° F.) to about 177° C. (350° F.), or about 79° C.(175° F.) to about 149° C. (300° F.), or about 93° C. (200° F.) to about135° C. (275° F.), or about 110° C. (230° F.) to about 121° C. (250°F.), or about 113° C. (235° F.) to about 118° C. (245° F.). Steam may bedirected through the column (at any suitable location, such as nearentry of the off-gas and/or the opposite end of the volatile impurityexit) to provide a sweeping vapor phase which may aid in the removal ofthe volatile impurity. In some embodiments, steam is directed throughthe column (e.g., through the first column).

Removal of Bio-Byproduct Impurity

In some of the embodiments described herein, the solution comprisingisoprene and bio-byproduct impurities (e.g., any isoprene-rich solutiontransferred from a first column) is transferred to a column (e.g., asecond column) wherein isoprene is stripped from the solution. In someof these embodiments, the stripping results in: an isoprene-leansolution comprising a major portion of the bio-byproduct impurity; and apurified isopene composition. In some embodiments, the second column isseparated from the first column. In some embodiments, the first andsecond columns are combined into one column (e.g., the functions of thefirst and second columns are combined into one column, such as a unitedtandem column wherein the solvent enters the first column at or near oneend and exits the second column at or near an opposite end).

In some embodiments, the temperature of the solution in the column(e.g., at the second column) is from about 66° C. (150° F.) to about177° C. (350° F.), or about 79° C. (175° F.) to about 149° C. (300° F.),or about 93° C. (200° F.) to about 135° C. (275° F.), or about 110° C.(230° F.) to about 121° C. (250° F.), or about 113° C. (235° F.) toabout 118° C. (245° F.). Steam may be directed through the column (atany suitable location, such as the opposite end of the entry point ofthe isoprene-rich solution and/or the near the isoprene-lean solutionexit) to provide a sweeping vapor phase which may aid in recovery of theisoprene from the solvent. In some embodiments, steam is directedthrough the column (e.g., through the second column).

In some embodiments, stripping the isoprene comprises increasing thepressure of the solution at the column (e.g., the second column). Insome of these embodiments, the solution comprising isoprene andbio-byproduct impurity (e.g., any isoprene-rich solution transferredfrom a first column) at the column (e.g., the second column), has apressure of more than any of about 5 PSIA, 10 PSIA, 20 PSIA, 30 PSIA, 40PSIA, 50 PSIA, 60 PSIA, 70 PSIA, 80 PSIA, 90 PSIA, 100 PSIA, 110 PSIA,120 PSIA, 130 PSIA, 140 PSIA, or 150 PSIA. In some embodiments, thepressure is any of about 5 PSIA to about 150 PSIA, about 5 PSIA to about100 PSIA, about 10 PSIA to about 75 PSIA, about 10 PSIA to about 65PSIA, about 10 PSIA to about 60 PSIA, about 15 PSIA to about 50 PSIA,about 15 PSIA to about 45 PSIA, about 15 PSIA to about 35 PSIA, or about15 PSIA to about 30 PSIA.

Additional Purification

The purified isoprene composition resulting from any of the methodsdescribed herein (e.g., a purified isopene composition stripped from thesecond column) may be further purified by any suitable means forinstance as shown in FIG. 1 with reference to adsorption system 36. Forexample, the purified isoprene composition may be further purified usingstandard techniques, such fractionation, additional gas stripping,adsorption/desorption, pervaporation, thermal or vacuum desorption ofisoprene from a solid phase, counter-current liquid-liquid extractionwith a suitable solvent, or extraction of isoprene immobilized orabsorbed to a solid phase with a solvent (see, for example, U.S. Pat.No. 4,703,007 and U.S. Pat. No. 4,570,029, which are each herebyincorporated by reference in their entireties, particularly with respectto isoprene recovery and purification methods). Suitable solventsinclude but are not limited to sodium hydroxide, sodium bicarbonate,sodium carbonate, potassium hydroxide, potassium bicarbonate, potassiumcarbonate, water, ionic liquids such as 1-ethyl-3-methylimidazoliumacetate, 1-ethyl-3-methylimidazolium ethyl sulfate, choline acetate,1-butyl-4-methylpyridinium tetrafluoroborate,1-hexyl-3-methylimidazolium chloride, 1-ethyl-3-methylimidazoliumthiocyanate, and 1-ethyl-3-methylimidazolium hydrogen sulfate.

Additional gas stripping involves the removal of isoprene vapor in acontinuous manner. Such removal can be achieved in several differentways including, but not limited to, adsorption to a solid phase,partition into a liquid phase, or direct condensation. In someembodiments, membrane enrichment of a dilute isoprene vapor stream abovethe dew point of the vapor resulting in the condensation of liquidisoprene. The additional purification of the purified isoprenecomposition may involve one step or multiple steps.

In some embodiments, the resulting isoprene of any of the methodsdescribed herein is further purified by treatment with an adsorptionsystem (e.g., an adsorption system comprising activated carbon, alumina,silica, and/or Selexsorb®.) Other suitable materials are zeolites andmolecular sieves, see U.S. Pat. Nos. 4,147,848; 5,035,794; and6,987,152. Suitable filter housings for such a system include those usedin the petrochemical industry for removal of impurities present in crudehydrocarbon streams. Examples include those supplied by The HilliardCorporation (Elmira, N.Y.) and ISC Corporation (Plano, Tex.) In someembodiments, the resulting isoprene of any of the methods describedherein is further purified by treatment with an adsorption systemcomprising silica. In some embodiments, the resulting isoprene of any ofthe methods described herein is further purified by distillation (e.g.,reflux condensation) before or after any other optional addedpurification. In some embodiments, the resulting isoprene of any of themethods described herein is further purified by treatment with anadsorption system and distillation (e.g., an adsorption systemcomprising silica and reflux condensation). Adsorption is typicallyconducted in a packed column configuration and is applicable to isopreneboth in vapor or liquid state. If isoprene is fed as a vapor, it iscommonly done by feeding it to the top of the column; on the other hand,if it is fed as a liquid, it is usually done by feeding to the bottom.Appropriate adsorbents include but are not limited to the following:activated carbon (e.g., NUCON G60, GC60, Vapor Filtration GC 4×8S, TIGG5CC 0408), activated alumina (e.g., Axens SAS 351, SAS 830, BASFSelexsorb CD), silica gel (Eagle Chemical Grade 148, Grade 140), and 3A,5A, or 13× molecular sieves.

Solvent Recirculation and Purification

In any of the methods described herein, the resulting solution followingisoprene stripping from the second column (e.g., the isoprene-leansolution comprising a major portion of the bio-byproduct impurity) maybe recycled back to the first column for reuse. In some embodiments,bio-byproduct is removed from the recycled solution prior to reuse(e.g., prior to reentering the first column). In some embodiments of anyof any method described herein, the method further comprises purifyingthe isoprene-lean solution to remove a major portion of thebio-byproduct impurity; and transferring the resulting solvent to thefirst column for reuse. In some embodiments, purifying the strippedsolution prior to reuse comprises treating the solution with anadsorption system (e.g., an adsorption system comprising activatedcarbon, alumina, silica, and/or Selexsorb®.) This absorption may also bedone using, e.g., a packed column. In some embodiments, purifying thestripped solution prior to reuse comprises treating the solution with asilica-based adsorption system. In some embodiments, purifying thestripped solution prior to reuse comprises liquid-liquid extraction. Insome embodiments, purifying the stripped solution prior to reusecomprises treating the solution comprises distillation. In someembodiments, purifying the stripped solution prior to reuse comprisestreating the solution with an adsorption system (e.g., a silica-basedadsorption system) and liquid-liquid extraction (in any order). In someembodiments, purifying the stripped solution prior to reuse comprisestreating the solution with an adsorption system (e.g., a silica-basedadsorption system), liquid-liquid extraction, and distillation (in anyorder). In any of these embodiments, the stripped solution (e.g., theisoprene-lean solution) may be purified by any of the described means(e.g., adsorption, liquid-liquid extraction, and/or distillation) suchthat the amount of bio-byproduct in the stripped solution is reduced bymore than any of about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 95% following purification. In some embodiments, the temperatureof the stripped solution (e.g., the isoprene-lean solution comprising amajor portion of the bio-byproduct impurity) is reduced prior to reusein the first column. In some embodiments, the stripped solution ispurified and the temperature is reduced prior to reuse in the firstcolumn. In some of these embodiments, the temperature is reduced priorto purification. In some of these embodiments, the temperature isreduced after purification. In some embodiments, the temperature of thestripped solution is reduced to less than any of about 95%, 90%, 80%,70%, 60%, 50%, 40%, 30%, 20%, or 10% of the temperature in ° F. from thesecond column prior to reuse (e.g., prior to reentering the firstcolumn). In some embodiments, the temperature stripped solution isreduced to less than any of about 121° C. (250° F.), 107° C. (225° F.),93° C. (200° F.), 79° C. (175° F.), 66° C. (150° F.), 52° C. (125° F.),38° C. (100° F.), 24° C. (75° F.), 10° C. (50° F.) or −4° C. (25° F.).In some embodiments, the temperature stripped solution is reduced to anyof about −4° C. (25° F.) to about 121° C. (250° F.), about −4° C. (25°F.) to about 79° C. (175° F.), about −4° C. (25° F.) to about 66° C.(150° F.), about −4° C. (25° F.) to about 38° C. (100° F.), or about −4°C. (25° F.) to about 24° C. (75° F.).

Recollection of Residual Isoprene from Vapor

In some instances, vapor removed from the first column (e.g., the vaporcomprising a major portion of the volatile impurity) may also comprisedesirable minor amounts of isoprene (e.g., residual isoprene notremaining in the isoprene-rich solution). In some of the embodimentsdescribed herein, the vapor comprising a major portion of the volatileimpurity additionally comprises a minor portion of isoprene. In any ofthe embodiments of the methods described herein, the method furthercomprises removing from vapor a minor portion of the isoprene, ifpresent. The residual isoprene may be recollected for use from the vaporcomprising a major portion of the volatile impurity by any suitablemeans (e.g., with an adsorption system). As described herein for furtherpurification of a purified isopene composition, any suitable technique,such as fractionation, additional gas stripping, adsorption/desorption,pervaporation, thermal or vacuum desorption of isoprene from a solidphase, or extraction of isoprene immobilized or absorbed to a solidphase with a solvent (see, for example, U.S. Pat. No. 4,703,007 and U.S.Pat. No. 4,570,029, which are each hereby incorporated by reference intheir entireties, particularly with respect to isoprene recovery andpurification methods) may be used to isolate residual isoprene from thevapor phase. As described, isoprene vapor can be removed in a continuousmanner, such as, but not limited to, adsorption to a solid phase,partition into a liquid phase, or direct condensation. Theremoval/purification of isoprene from the vapor phase may involve onestep or multiple steps. In any of the embodiments of the methodsdescribed herein, the method further comprises removing isoprene (ifpresent) from the vapor, with an adsorption system (e.g., an adsorptionsystem comprises activated carbon, alumina, silica, and/or Selexsorb®).In any of the embodiments of the methods described herein, the methodfurther comprises removing isoprene (if present) from the vapor with anactivated carbon adsorption system.

Capture Device

The methods described herein may optionally use a capture device (suchas system 38 in FIG. 1) capable of reducing the total amount ofundesirable components released into the atmosphere (e.g., CO₂) from thevapor. A generic carbon-based adsorption unit such as those used forsolvent recovery and supplied by manufacturers including AMCEC Inc.(Lisle, Ill.) and Nucon International Inc. (Columbus, Ohio) would besuitable.

It is often desirable to capture the trace amount of isoprene or othercomponents in the fermentation off-gas that is not recovered by theprimary process both for value of the product as well as minimizingrelease of undesirable components such as carbon dioxide to environment.Trace levels of isoprene and high molecular weight organic compounds canbe effectively captured by adsorption on solid surface such as activatedcarbon (e.g., see NUCON G60, GC60, Vapor Filtration GC 4×85, TIGG 5CC0408). Carbon dioxide capturing is commonly carried out in acounter-current gas scrubber/absorber where the scrubbing fluid is fedto the top of the liquid contactor while the gas being scrubbed is fedto the bottom. The liquid contactor will have sufficient contact surfaceor equilibrium stages to achieve the desired reduction in concentration.Common scrubbing fluids include but are not limited to monoethanolamine(MEA), piperazine, water or a combination of all (see, e.g., CO₂Absorption Rate and Solubility in Monoethanolamine/piperazine/Water,Hongyi Dang, et al., Prepared for presentation at the First NationalConference on Carbon Sequestration, Washington, D.C., May 14-17, 2001).

Resulting Compositions

In some aspects, the methods described herein provide a purifiedisoprene composition, wherein a purified isoprene composition is anisoprene composition that has been separated from at least a portion ofone or more components that are present in the fermentor off-gas. Insome embodiments, the purified isoprene composition has a purity ofgreater than about 75% (w/w). In some embodiments, the purified isoprenecomposition has a purity of greater than any of about 80%, 85%, 90%,95%, 97%, 98%, 99%, 99.5%, 99.5%, or 99.95% (w/w).

In any of the embodiments described herein, the purified isoprenecomposition comprises no more than about 20% (w/w) bio-byproductimpurity. In some embodiments, the purified isoprene compositioncomprises less than about 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, 1%,0.075%, 0.5%, 0.25%, 0.1%, 0.05%, 0.02%, 0.01%, or 0.005% (w/w)bio-byproduct impurity relative to the weight of the isoprene. In someembodiments, the purified isoprene composition comprises less than about50% (w/w) bio-byproduct impurity relative to the bio-byproduct impurityof the fermentor off-gas. In some embodiments, the purified isoprenecomposition comprises less than any of about 40%, 35%, 30%, 25%, 20%,15%, 10%, 7.5%, 5%, 2.5%, 1%, or 0.5% (w/w) bio-byproduct impurityrelative to the bio-byproduct impurity of the fermentor off-gas.

In any of the embodiments described herein, the purified isoprenecomposition comprises no more than about 20% (w/w) volatile impurity. Insome embodiments, the purified isoprene composition comprises less thanabout 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, 1%, 0.075%, 0.5%, 0.25%,0.1%, or 0.05% (w/w) volatile impurity. In some embodiments, thepurified isoprene composition comprises less than about 50% (w/w)volatile impurity relative to the volatile impurity of the fermentoroff-gas. In some embodiments, the purified isoprene compositioncomprises less than any of about 40%, 35%, 30%, 25%, 20%, 15%, 10%,7.5%, 5%, 2.5%, 1%, or 0.5% (w/w) volatile impurity relative to thevolatile impurity of the fermentor off-gas.

In any of the embodiments described herein, the purified isoprenecomposition comprises no more than about 20% (w/w) of a one or morecompounds selected from H₂O, CO₂, CO, N₂, CH₄, H₂ and O₂. In someembodiments, the purified isoprene composition comprises no more thanabout 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, 1%, 0.075%, 0.5%, 0.25%,0.1%, or 0.05% (w/w) of one or more compounds selected from H₂O, CO₂,CO, N₂, CH₄, H₂ and O₂. In some embodiments, the purified isoprenecomposition comprises less than about 50% (w/w) of one or more compoundsselected from H₂O, CO₂, CO, N₂, CH₄, H₂ and O₂ relative to the fermentoroff-gas. In some embodiments, the purified isoprene compositioncomprises less than any of about 40%, 35%, 30%, 25%, 20%, 15%, 10%,7.5%, 5%, 2.5%, 1%, or 0.5% (w/w) one or more compounds selected fromH₂O, CO₂, CO, N₂, CH₄, H₂ and O₂ relative to the fermentor off-gas.

In any of the embodiments described herein, the purified isoprenecomposition comprises no more than about 20% (w/w) CO₂. In someembodiments, the purified isoprene composition comprises no more thanabout 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, 1%, 0.075%, 0.5%, 0.25%,0.1%, or 0.05% (w/w) CO₂. In some embodiments, the purified isoprenecomposition comprises less than about 50% (w/w) CO₂ relative to theamount of CO₂ of the fermentor off-gas. In some embodiments, thepurified isoprene composition comprises less than any of about 40%, 35%,30%, 25%, 20%, 15%, 10%, 7.5%, 5%, 2.5%, 1%, or 0.5% (w/w) CO₂ relativeto the amount of CO₂ of the fermentor off-gas.

In any of the embodiments described herein, the purified isoprenecomposition comprises no more than about 20% (w/w) O₂. In someembodiments, the purified isoprene composition comprises no more thanabout 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, 1%, 0.075%, 0.5%, 0.25%,0.1%, or 0.05% (w/w) O₂. In some embodiments, the purified isoprenecomposition comprises less than about 50% (w/w) O₂ relative to theamount of O₂ of the fermentor off-gas. In some embodiments, the purifiedisoprene composition comprises less than any of about 40%, 35%, 30%,25%, 20%, 15%, 10%, 7.5%, 5%, 2.5%, 1%, or 0.5% (w/w) O₂ relative to theamount of O₂ of the fermentor off-gas.

Isoprene Compositions

Also provided are purified isoprene compositions (e.g., compositionscomprising purified bioisoprene). In some embodiments is provided apurified isopene composition preparable by any of the methods describedherein. In some embodiments, is provided a purified isoprene compositionprepared by any of the methods described here.

In some embodiments, there is provided a composition of isoprene (e.g.,bioisoprene) comprising less than about 15%, 12%, 10%, 8%, 6%, 5%, 4%,3%, 2%, 1%, 0.075%, 0.5%, 0.25%, 0.1%, 0.05%, 0.02%, 0.01%, or 0.005%(w/w) bio-byproduct impurity relative to the weight of the isoprene. Insome embodiments, is provided a composition of isoprene (e.g.,bioisoprene) comprising less than about 15%, 12%, 10%, 8%, 6%, 5%, 4%,3%, 2%, 1%, 0.075%, 0.5%, 0.25%, 0.1%, or 0.05% (w/w) volatile impurity.In some embodiments, is provided a composition of isoprene (e.g.,bioisoprene) comprising less than about 15%, 12%, 10%, 8%, 6%, 5%, 4%,3%, 2%, 1%, 0.075%, 0.5%, 0.25%, 0.1%, 0.05%, 0.02%, 0.01%, or 0.005%(w/w) bio-byproduct impurity relative to the weight of the isoprene andless than about 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, 1%, 0.075%, 0.5%,0.25%, 0.1%, or 0.05% (w/w) volatile impurity relative to the weight ofthe composition. In any of these embodiments, the isoprene compositioncomprises greater than any of about 80%, 85%, 90%, 95%, 97%, 98%, 99%,99.5%, 99.5%, or 99.95% (w/w) isoprene relative to the weight of thecomposition. In any of these embodiments, the isoprene compositioncomprises greater than about 99.94%, 99.94%, 99.95%, 99.96%, 99.97%,99.98%, or 99.99% isoprene (w/w) relative to the weight of all C5hydrocarbons. In any of these compositions, the bio-byproduct impuritycomprises one or more compounds selected from the group consisting of2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine,2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methylacetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethylacetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone,1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate,3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate,3-methyl-3-but-1-enyl acetate, 3-methyl-2-but-1-enyl acetate,(E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene,and 2,3-cycloheptenolpyridine or as indicated above.

In some embodiments, is provided a composition of isoprene (e.g.,bioisoprene) comprising less than about 5% (or 1%, or 0.5%, or 0.05%, or0.005%) (w/w) bio-byproduct impurity relative to the weight of theisoprene; less than about 10% (or 1%, or 0.1%, or 0.05%) (w/w) volatileimpurity relative to the weight of the composition; and greater thanabout 95% (or 98%, or 99%, or 99.95%) (w/w) isoprene relative to theweight of the composition, wherein the isoprene composition comprisesgreater than about 99.9% (or 99.95%, or 99.97%, or 99.99%) isoprene(w/w) relative to the weight of all C5 hydrocarbons. In someembodiments, is provided a composition of isoprene comprising less thanabout 1% (w/w) bio-byproduct impurity relative to the weight of theisoprene; less than about 5% (w/w) volatile impurity relative to theweight of the composition; and greater than about 98% (w/w) isoprenerelative to the weight of the composition, wherein the isoprenecomposition comprises greater than about 99.95% isoprene (w/w) relativeto the weight of all C5 hydrocarbons. In some embodiments, is provided acomposition of isoprene comprising less than about 1% (w/w)bio-byproduct impurity relative to the weight of the isoprene; less thanabout 5% (or 2%, or 1%, or 0.5%) CO₂ (w/w) relative to the weight of thecomposition; and greater than about 98% (w/w) isoprene relative to theweight of the composition, wherein the isoprene composition comprisesgreater than about 99.95% isoprene (w/w) relative to the weight of allC5 hydrocarbons.

In some embodiments of any of the compositions, at least a portion ofthe isoprene is in a gas phase. In some embodiments, at least a portionof the isoprene is in a liquid phase (such as a condensate). In someembodiments, at least a portion of the isoprene is in a solid phase. Insome embodiments, at least a portion of the isoprene is adsorbed to asolid support, such as a support that includes silica and/or activatedcarbon.

In any of the compositions described herein, the composition maycomprise greater than about 2 mg of isoprene, such as greater than orabout 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000 mg of isoprene. In some embodiments, thecomposition comprises greater than or about 2, 5, 10, 20, 30, 40, 50,60, 70, 80, 90, 100 g of isoprene. In some embodiments, the amount ofisoprene in the composition is between about 2 to about 5,000 mg, suchas between about 2 to about 100 mg, about 100 to about 500 mg, about 500to about 1,000 mg, about 1,000 to about 2,000 mg, or about 2,000 toabout 5,000 mg. In some embodiments, the amount of isoprene in thecomposition is between about 20 to about 5,000 mg, about 100 to about5,000 mg, about 200 to about 2,000 mg, about 200 to about 1,000 mg,about 300 to about 1,000 mg, or about 400 to about 1,000 mg.

In some embodiments, the composition includes ethanol. In someembodiments, the composition includes between about 75 to about 90% byweight of ethanol, such as between about 75 to about 80%, about 80 toabout 85%, or about 85 to about 90% by weight of ethanol. In someembodiments in which the composition includes ethanol, the compositionalso includes between about 4 to about 15% by weight of isoprene, suchas between about 4 to about 8%, about 8 to about 12%, or about 12 toabout 15% by weight of isoprene.

Additional methods and compositions are described in as described inInternational Patent Application Publication No. WO2009/076676; U.S.patent application Ser. Nos. 12/496,573, 12/560,390, 12/560,317,12/560,370, 12/560,305, and 12/560,366; and U.S. provisional patentapplication Nos. 61/187,930, 61/187,934, and 61/187,959, all of whichare incorporated by reference in their entireties, particularly withrespect to compositions and methods for producing isoprene.

This invention is illustrated by the following examples that are merelyfor the purpose of illustration and are not to be regarded as limitingthe scope of the invention or the manner in which it can be practiced.Unless specifically indicated otherwise, parts and percentages are givenby weight.

EXAMPLES

Example 1 is recovery of isoprene from fermentation off-gas byabsorption and stripping, including:

(1) Absorption of isoprene from fermentation off-gas. Fermentationoff-gas comprising isoprene, bio-byproduct impurities and volatileimpurities is introduced into a glass gas scrubber unit (Part #CG-1830-10, supplied by ChemGlass, Vineland, N.J., USA) at a flow rateof 4 L/min. The reservoir of the scrubber contains 0.5 L of Isopar® M(ExxonMobil, Tex.) which is recirculated at a rate of 2 L/min. Thesolvent is recirculated until equilibrium with the fermentation off-gasis achieved, as determined by GC/MS analysis of the fermentation off-gasprior to entering the gas scrubber unit, the isoprene-rich Isoparsolvent and the tail-gas emerging from the gas scrubber unit.Equilibrium occurs at the point where the isoprene concentration in thefeed gas is the same as that in the tail gas emerging from the scrubber.Another indication is the point at which the isoprene-concentration inthe solvent attains a steady state.

(2) Stripping and condensation of isoprene. Stripping of isoprene fromthe isoprene-rich Isopar solvent is achieved by reconfiguring the gasscrubber unit, whereby steam is added to the gas scrubber unit in placeof the fermentation off-gas feed at a rate of 4 L/min. The solvent isrecirculated at a rate of 2 L/min and the isoprene vapor stripped fromthe solvent emerges from the top of the gas scrubber unit, along withamounts of bio-byproduct impurities.

The isoprene vapor emerging from the gas scrubber unit is then condensedusing a Graham condenser or similar glass condenser cooled with acoolant at 0 to 10° C. The isoprene condensate is collected andinhibited through the addition of 150 ppm t-butylcatechol. The purity ofthe liquid isoprene is determined by GC/MS according to procedures knownto those familiar in the art.

The following describes two exemplary sets of columns for use here,suitable for large scale (manufacturing) or smaller scale (a pilot plantor test apparatus) processes as determined by simulation.

Example A below uses tray columns, there being thirteen trays for theabsorber column 14 and sixteen trays for the stripper column 24. ExampleB below uses structured packing columns, there being ten stages (trayequivalents) for the absorber column 14 and eleven stages for thestripper column 24. The goal is 99.9% recovery of the isoprene. Allthese parameters and these examples are merely illustrative.

Solvent % Recovery gpm/MSCFH of Contained gas feed Isoprene Example AFermentor Gas 0.38 89.0% Isoprene 0.40 91.0% Concentration: 0.41 94.0%0.12 mole fraction 0.43 97.5% 0.21 wt. fraction 0.47 99.9% Lbs.Stripping Steam/Lb. 1.10 Recovered Isoprene @ 99.9% Recovery RecoveredIsoprene Purity 99.8 Wt % Example B Fermentor Gas 0.28 89.0% Isoprene0.29 91.0% Concentration: 0.31 94.0% 0.04 mole fraction 0.33 97.5% 0.08wt. fraction 0.41 99.9% Lbs. Stripping Steam/Lb. 2.79 Recovered Isoprene@ 99.9% Recovery Recovered Isoprene Purity 99.8 Wt %

Example 2 is of recovery of isoprene from fermentation off-gas usingsolvent, by a laboratory scale gas scrubber unit as described above.

Fermentation off-gas including isoprene, bio-byproduct impurities andvolatile impurities was introduced into a laboratory-type glass gasscrubber unit including an absorption column (Part # CG-1830-10,supplied by ChemGlass, Vineland, N.J., USA), at a flow rate of 8 L/min.The isoprene concentration was in the range of 1.8 to 2.1% v/v asdetermined by online mass spectrometry using a Hiden HPR-20 massspectrometer (supplied by Hiden Analytical, United Kingdom). Thereservoir of the scrubber contained 1 L of Isopar®L isoparaffinicsolvent as described above, hereinafter “solvent” (supplied byExxonMobil Chemical Co., Houston, Tex., USA) which was recirculated at arate of 2 L/min at room temperature (20° C.). The concentration ofisoprene in the Isopar solution was about 1% by volume during thisprocess. The process was continued until equilibrium with thefermentation off-gas was achieved, as determined by online massspectrometer analysis of the fermentation off-gas prior to entering thegas scrubber unit and the tail-gas emerging from the gas scrubber unit.These data were used to calculate the absorption efficiency of isoprene(vertical axis) as a function of time (horizontal axis), as shown in theplot of FIG. 2.

The cumulative amount of isoprene collected was calculated bymultiplying by the total productivity of isoprene by the averageabsorption efficiency over the duration of the process, as determined bythe extrapolated area under the plot of FIG. 2. At an isopreneconcentration of 2% v/v and an off-gas flow of 8 L/min, the total amountof isoprene produced by the fermentor over the 1.6 hour period wasapproximately 40 g, of which around 30% was collected, giving atheoretical concentration in the range of 10 to 12 g/L isoprene in thesolvent. Following the completion of the process, the solution wasremoved from the gas scrubber for subsequent analysis, stripping, andcondensation to recover pure isoprene liquid.

Example 3 is of an analysis of isoprene solution.

The isoprene solution generated by the above described gas absorptionwas analyzed to determine the isoprene content and the identity of majorimpurities using both headspace and liquid GC/MS (gaschromatography/mass spectrometry) methods. Isoprene concentration wasdetermined using a headspace method, whereby 1 mL of the isoprenesolution was placed into a 20 mL head space vial and incubated at 40° C.for 5 minutes prior to a 100 μL headspace injection. The GC/MS methodused helium as the carrier gas at 1 mL/min, an inlet temperature of 230°C. and a split ratio of 100:1. A Zebron TM ZB-5 GC column (30 m×0.25mm×0.25 μm and supplied by Phenomenex, Torrance, Calif., USA) wasemployed, with the mass spectrometer detector operating in SIM modemonitoring ions at m/z 41, 56, 68, 69, 71 and 86. The heating began at50° C., held for 2 minutes, followed by an increase to 75° C. at a rateof 20° C./min, then increasing to 250° C. at a rate of 35° C./min. Thefinal temperature of 250° C. was held for 0.75 minutes for a total runtime of 9 minutes. Under these conditions, isoprene eluted at 1.68minutes and solvent L-derived hydrocarbons eluted between 5.5 and 6.5minutes. The method was calibrated using isoprene/solvent standardsranging in concentration from 1 mg/mL to 20 mg/mL. The concentration ofthe isoprene/solvent composition generated in Example 2 was determinedto be 9.4 g/L using this method.

For identification of bio-byproduct impurities present in the isoprenesolution, a liquid GC/MS method was employed whereby a 1 μL sample wasinjected into a GC inlet held at 250° C. with a 20:1 split utilizinghelium as the carrier gas at a flow rate of 1 mL/min. The Zebron ZB-5 GCcolumn (30 m×0.25 mm×0.25 um) was employed, with the mass spectrometerdetector operating in scan mode monitoring ions between m/z 29 and 350.The heating began at 50° C., held for 2 minutes, followed by an increaseto 320° C. at a rate of 20° C./min with a final hold time of 2.5 minutesfor a total run time of 18 minutes. Under these conditions as shown inFIG. 3, isoprene eluted at 1.69 minutes (horizontal axis) andsolvent-derived hydrocarbons eluted between 5.5 and 9 minutes. Severalbio-byproduct impurities were identified (see Table 2), in addition tolow molecular weight saturated hydrocarbons derived from the solvent.Note that some of these impurities themselves are of commercial valueand could be further isolated as bio-byproducts using well known methodsin an industrial scale version of the present purification process.

TABLE 2 Compound Retention time (min) Ethanol 1.59 Acetone 2.653-methyl-3-buten-1-ol 3.02 3-methyl-2-buten-1-ol 3.483-methyl-2-buten-1-yl acetate 4.69

FIG. 4 is an expansion of the left hand portion GC/MS spectrum of FIG. 3from 1.6 minutes to 4.8 minutes (horizontal axis).

Example 4 is of stripping and condensation of isoprene liquid fromisoprene/solvent solutions.

Two methods (referred to above) were used to recover isoprene liquidfrom isoprene/solvent solutions generated as described in Example 2:

(a) In a laboratory scale process, stripping of the isoprene from thesolvent was achieved by transferring the isoprene/solvent solution to a3-necked 1 L round bottom flask fitted with a laboratory-type dry-icecooled Dewar-style distillation head (Part #CG-1251, supplied byChemglass, Vineland, N.J., USA), a gas sparge inlet and a stirrer bar.The condenser was fitted with a 50 mL receiving flask for the liquidisoprene product. The outlet from the apparatus was sent to a dry-icefilled cold trap and a bubbler to monitor gas flow. The flask was heatedto 80° C. in an oil bath and nitrogen gas bubbled through the solution arate of less than 1 L/minute. Over the course of 2 hours, liquidisoprene (about 4 mL) was collected in the receiving flask.

(b) The apparatus described in (a) above was modified by coupling a3-stage Snyder distillation column coupled between the 3-neck flask andthe condenser. The temperature of the oil bath was raised to 120° C. Inthis case, steam was used instead of nitrogen gas, the flow of which wasadjusted to maintain a temperature gradient in the distillation columnranging from 100° C. at the bottom to 34° C. at the top. Over the courseof 2 hours, liquid isoprene (about 6 mL) was collected in the receivingflask.

Analysis of solvent following this distillation was performed using theheadspace GC/MS method described in Example 3 in order to determine theextent to which isoprene was stripped from the solvent. The results areshown in Table 3:

TABLE 3 Stripping Isoprene concentration in solvent (g/L) Strippingmethod Initial Final Efficiency Nitrogen 8.55 5.52 41% Steam 9.38 3.8455%

Example 5 is an analysis of isoprene liquid recovered byabsorption/stripping using solvent.

The isoprene liquid generated as described in Example 4 was analyzedusing GC/FID (gas chromatography/flame ionization detector) and GC/MSmethods to assess overall purity and to identify both bio-byproduct andother impurities present. The GC/FID analysis was performed using aDB-Petro column (100 m×0.25 mm, 0.50 um film thickness supplied byAgilent Technologies, Santa Clara, Calif., USA) held at 50° C. for 15minutes. The method utilized helium as the carrier gas at a flow of 1mL/min. The injection port was held at 200° C. and operated in splitlessmode. An Agilent 5793N mass selective detector was fun in full scan modefrom m/z 19 to m/z 250. FIG. 5 is a GC/FID plot of isoprene recoveredfrom the solvent in this example. Under these conditions, isoprene wasobserved to elute at 13.4 min, and bio-byproduct impurities and volatilesolvent derived impurities between 12.6 and 23.0 minutes. Solventhydrocarbons eluted between 27 and 29.5 minutes.

Example 6 is of removal of polar bio-byproduct impurities from thesolvent in a final purification process as referred to above.

Polar bio-byproduct impurities present in the isoprene-solvent wereremoved by passage over an adsorbent as described above, in particularadsorbents based on silica and alumina. For example, the solventsolution (100 mL) obtained following stripping of the isoprene (seeExample 4) was pumped through a bed of Selexsorb CDX adsorbent (10 g ofSelexsorb CDX from BASF) over 20 minutes and filtered solvent analyzedby GC/FID. The chromatogram (not shown) showed that the majority ofbio-byproduct impurities were removed. As an alternative (see below), asilica based adsorbent may be used.

FIG. 6 shows an example of an adsorbent process apparatus (such assystem 36 in FIG. 1) where the isoprene solution output from theupstream portion of the FIG. 1 apparatus is initially held in feed tank(reservoir) 80. A flow of nitrogen gas is provided via flow line 82 toreservoir 80 to maintain a pressure of about 90 PSI (pounds per squareinch, about 6 atmospheres measured at pressure regulator P), via valveV1. Valve V2 admits the pressurized isoprene solution to valves V3, V4between which is coupled a pump 90. The pumped solution is carried bytransfer line 94 via valve V5 to a conventional adsorbing bed 98, whichis a bed of alumina, or silica or other adsorbent as described abovesuch as Selexsorb CDX, housed in a conventional jacket 100.

A second flow of nitrogen gas is provided via valves V8 and V6 withintervening rotameter 104 to measure the gas flow rate. This second flowof nitrogen gas is coupled to the bed 98. As is conventional, thenitrogen gas at value V6 and the isoprene solution at valve V5 aresupplied alternately to allow flushing via the nitrogen gas of theadsorbent in the bed 98. The flushing removes the impurities in theisoprene solution which have been adsorbed by the bed. This processallows the impurities to be vented with the flushing nitrogen gas viavalve V5 during this regeneration of the bed. Valves V9, V10 couple achiller/heater unit 108 to the bed 98 to keep both the flushing nitrogengas and the isoprene solution at their proper temperatures. Finally, theresulting purified isoprene solution is output via transfer line 110(having a second pressure regulator P) and valve V7.

In a laboratory scale example, isoprene derived from a bioisoprenecomposition (1 mL with 150 ppm TBC added) was treated with one bead(diameter ⅛″ which is 3 mm, about 90 mg by weight) of either Selexsorb®CD, or Selexsorb® CDX in a GC vial for 1 hour with occasional agitation.The Selexsorb® products changed color from white to yellowish within 10minutes. Samples were analyzed by gas chromatography/mass spectrometryand the spectra overlaid to highlight the degree to which impuritieswere removed. The extent of polar impurity removal was determined andthe results shown in Table 3A.

TABLE 3A Compound Selexsorb ® CD Selexsorb ® CDX Ethanol >90% >90%Acetone >90% >90% Methacrolein >90% >90% Ethyl acetate >90% >90%3-Methyl-3-buten-2-ol >90% >90% Methylvinyl ketone >90% >90%2-vinyl-2-methyloxirane >90% >90% 3-methyl-3-buten-1-ol  94%  96%3-methyl-3-buten-1-yl acetate  68%  75%Further Isoprene Purification—Liquid Extraction

As explained above and as depicted in FIG. 6, it is desirable to furtherpurify the isoprene solution, which typically contains a number ofimpurities of various types. In one embodiment, further purification wasachieved using the liquid extraction method referred to above, to removesemi-polar impurities.

It has been determined that a significant difference betweenconventional isoprene derived from petroleum and the present bioisoprenederived from fermentation is the presence in the fermentation-typeisoprene of large amounts of biological (“bio”) by-products that arepolar in nature in terms of their chemistry. These impurities fall intochemical classes including acetates, alcohols, ketones, and acids asdescribed above. These impurities interfere or inhibit the subsequentnecessary polymerization of the isoprene as described above andtherefore must be removed from the recovered isoprene prior to thedownstream polymerization step. The adsorbent process described withreference to FIG. 6 generally would not remove such polar impurities.

It has been found that contacting the bioisoprene with de-ionized (DI)water or a base (alkaline) de-ionized water solution removed asignificant amount of such impurities. Multiple contacts with the wateror alkaline water solution will reduce impurities to any desirablelevel. In yet another example, Table 4 shows (left hand column) thevarious impurities, the proportion of the impurity removed in thisexample with contact of an equal volume of a alkaline water solution(center column), and (right hand column) the proportion of the impurityremoved by contact with an equal volume of deionized water.

TABLE 4 % Removed (contact % Removed (contact Impurity with equal volumewith equal volume (0.1M in n-hexane) of (10) wt % NaOH) of DI water)2-Methylfuran 2.6% 0.4% Methanol 100.0% 100.0% Prenol 55.7% 18.3%Acetone 71.2% 90.1% Acetic Acid Co-elute with hexane Methyl Isobutyrate89.3% 9.1% Methyl Acetate 38.3% 24.2% Dimethyl Bisulfide 4.4% 2.6%Thus it has been found that the base (alkaline) water process in thesecond column of Table 4 was effective in removing all these impuritiesto a large extent, except for the 2-methylfuran and the dimethyldisulfide. Only a small proportion of these two impurities was removed.However, it has been found that the 2-methylfuran is not significant interms of preventing polymerization. Hence dimethyl sulfide is the keyremaining impurity. It is well known that dimethyl disulfide is aparticularly potent polymerization “poison.”

An apparatus to perform this “caustic wash” process is conventional andwould include a suitable vessel to hold the caustic solution and intowhich a volume of the isoprene solution is pumped. The vessel would beequipped with a suitable stirring or mixing device, since the isoprenesolution is not miscible with water. Hence the above reference to“contacting” the isoprene solution with the caustic solution long enoughto achieve the desired extraction of the impurities into the causticsolution. Then the caustic solution is separated conventionally from thepurified isoprene solution. This caustic wash process may be aconventional batch or continuous process. This caustic wash process maybe performed upstream or downstream of the FIG. 6 adsorption system. Or,the caustic wash and adsorption may be performed together by, e.g.,impregnating the silica or alumina adsorbent with a suitable causticcompound, as is known in the field.

Further, an effective way of removing the dimethyl disulfide is theadsorption technique as described above with reference to FIG. 6. FIG. 7is a plot of impurities in bioisoprene over time (horizontal axis) wherethe right hand peak shows the dimethyl disulfide concentration after anelution time (in a laboratory type adsorbent bed purifying apparatus)indicated along the horizontal axis. As seen, the initial concentrationof dimethyl disulfide was quite high then fell substantially whentreated with an adsorption system having alumina, and fell even furtherwhen treated with the silica adsorption, to be almost imperceptible.

FIG. 8 also refers to this adsorbent technique and shows time along thehorizontal axis, and along the left hand vertical axis and associatedleft hand plot the proportion of dimethyl disulfide in the silica as apercentage of the silica, and along the right hand vertical axis andassociated right hand plot the percentage of dimethyl disulfide in thefeed that is not absorbed by the silica over time. Therefore theeffectiveness of the adsorption process diminished sharply beginning atabout 60 minutes as the adsorbent bed became loaded (saturated) with thedimethyl disulfide. Per the left hand plot, the bed becomes saturated atabout 38% dimethyl disulfide. Hence the need to periodically flush(regenerate) the bed as described with reference to FIG. 6.

Further FIG. 9 shows, in terms of relative concentrations, the presenceof nitrogen, isoprene and dimethyl disulfide in the isoprene solution atvarious times during the adsorption technique, showing the same effectas FIG. 8. This illustrates the initial gas composition (pre-treatment)at the lowest plot proceeding to the end of a cycle of the process atthe upper plot, that the dimethyl disulfide was essentially eliminatedwhile the amounts of the other two compounds, which are the dissolvednitrogen and the isoprene, were essentially the same. Note that at 120minutes the dimethyl disulfide peak reappears when the saturated bedallows the dimethyl disulfide to “break through.”

The above examples, which are intended to be purely exemplary of theinvention and should therefore not be considered to limit the inventionin any way, also describe and detail aspects and embodiments of theinvention discussed above. Unless indicated otherwise, temperature is indegrees Centigrade and pressure is at or near atmospheric pressure. Theforegoing examples and detailed description are offered by way ofillustration and not by way of limitation. All publications, patentapplications, and patents cited in this specification are hereinincorporated by reference as if each individual publication, patentapplication, or patent were specifically and individually indicated tobe incorporated by reference. In particular, all publications citedherein are expressly incorporated herein by reference for the purpose ofdescribing and disclosing compositions and methodologies which might beused in connection with the invention. Although the foregoing inventionhas been described in some detail by way of illustration and example forpurposes of clarity of understanding, it will be readily apparent tothose of ordinary skill in the art in light of the teachings of thisinvention that certain changes and modifications may be made theretowithout departing from the spirit or scope of the appended claims.

What is claimed is:
 1. A method of purifying isoprene from a fermentoroff-gas comprising: contacting a fermentor off-gas, wherein the off-gascomprises a bioisoprene composition comprising isoprene, volatileimpurity, and bio-byproduct impurity, with a solvent in a first columnto form: an isoprene-rich solution comprising the solvent, a majorportion of the isoprene and a major portion of the bio-byproductimpurity present in the off-gas; and a vapor comprising a portion of thevolatile impurity present in the off-gas; transferring the isoprene-richsolution from the first column to a second column; and strippingisoprene from the isoprene-rich solution in the second column to form:an isoprene-lean solution comprising a portion of the bio-byproductimpurity present in the off-gas; and a purified isoprene composition. 2.The method of claim 1 wherein the volatile impurity comprises a compoundselected from the group consisting of H₂O, CO₂, N₂, H₂, CO and O₂. 3.The method of claim 1, wherein the bio-byproduct impurity comprises acompound selected from the group consisting of ethanol, acetone,methanol, acetaldehyde, methacrolein, methyl vinyl ketone,3-methylfuran, 2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene,a C5 prenyl alcohol (such as 3-methyl-3-buten-1-ol or3-methyl-2-buten-1-ol), 2-heptanone, 6-methyl-5-hepten-2-one,2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal,methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol,3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone,3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butylacetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate,3-methyl-2-buten-1-yl acetate, (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, (E,E)-3,7,11-trimethyl-1,3,6,10-dodecatetraene,(E)-7,11-dimethyl-3-methylene-1,6,10-dodecatriene,3-hexen-1-ol,3-hexen-1-ylacetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol), andcitronellol (3,7-dimethyl-6-octen-1-ol).
 4. The method of claim 1,wherein the solvent has a boiling point of greater than about 177° C. 5.The method of claim 1, wherein the solvent has a CO₂ Ostwald coefficientat 54° C. of less than about 1.25.
 6. The method of claim 1, wherein thesolvent has a Kauri-butanol value of less than about
 50. 7. The methodof claim 1, wherein the solvent has an Aniline Point of greater thanabout 66° C.
 8. The method of claim 1, wherein the solvent has akinematic viscosity at 40° C. is less than about 2.5 centistokes (cSt).9. The method of claim 1, wherein the solvent has a surface tension at25° C. from about 20 to 30 dyne/cm.
 10. The method of claim 1, whereinthe solvent has an average molecular weight from about 125 to about 225u.
 11. The method of claim 1 wherein the solvent is an isoparaffin or aparaffin.
 12. The method claim 1, wherein the solvent further comprisesa polymerization inhibitor.
 13. The method of claim 1, furthercomprising reducing the temperature of the fermentor off-gas prior tocontacting the solvent in the first column.
 14. The method of claim 1,further comprising transferring the fermentor off-gas to an isolationunit, thereby stabilizing the off-gas pressure, prior to contacting thefermentor off-gas with the solvent in the first column.
 15. The methodof claim 1, further comprising at least partially condensing thefermentor off-gas prior to contacting the solvent in the first column.16. The method of claim 1, wherein the contacting the fermentor off-gaswith a solvent in a first column comprises supplying stripping vaporfrom a bottom of the first column.
 17. The method of claim 1, whereinthe contacting the fermentor off-gas with a solvent in a first columnfurther comprises adding steam to the first column.
 18. The method ofclaim 1, wherein the stripping isoprene from the isoprene-rich solutionin the second column comprises adding steam to the second column. 19.The method of claim 1, further comprising: purifying the isoprene-leansolution to remove a major portion of the bio-byproduct impurity; andtransferring the isoprene-lean solution to the first column for reuse.20. The method of claim 19, wherein purifying the isoprene-lean solutioncomprises treating the isoprene-lean solution with an adsorption system.21. The method of claim 19, wherein purifying the isoprene-lean solutioncomprises distillation.
 22. The method of claim 1, further comprisingreducing the temperature of the isoprene-lean solution prior to removinga major portion of the bio-byproduct impurity.
 23. The method of claim1, comprising further purifying the purified isoprene composition. 24.The method of claim 23, wherein further purifying the purified isoprenecomposition comprises distillation.
 25. The method of claim 23, whereinfurther purifying the purified isoprene composition comprises treatingthe purified isoprene composition with an adsorption system.
 26. Themethod of claim 1, further comprising removing from the vapor a portionof the isoprene.
 27. The method of claim 1, wherein the fermentoroff-gas is provided to the first column at a pressure greater thanatmospheric.
 28. The method of claim 1, wherein the purified isoprenecomposition has a purity greater than about 90%.
 29. The method of claim1, wherein the purified isoprene composition comprises less than about25% bio-byproduct impurity relative to the amount of bio-byproductimpurity of the fermentor off-gas.
 30. The method of claim 1, whereinthe purified isoprene composition comprises less than about 25% volatileimpurity relative to the amount of volatile impurity of the fermentoroff-gas.
 31. A purified isoprene composition prepared by the method ofclaim
 1. 32. The method of claim 23, wherein the further purifyingcomprises contacting the purified isoprene composition with water or abase and water.
 33. A method of purifying isoprene from a fermentoroff-gas comprising: contacting a fermentor off-gas comprising isoprene,volatile impurity, and bio-byproduct impurity, with a solvent in a firstcolumn to form: an isoprene-rich solution comprising the solvent, amajor portion of the isoprene and a major portion of the bio-byproductimpurity present in the off-gas; and a vapor comprising a portion of thevolatile impurity present in the off-gas; transferring the isoprene-richsolution from the first column to a second column; and strippingisoprene from the isoprene-rich solution in the second column to form:an isoprene-lean solution comprising a portion of the bio-byproductimpurity present in the off-gas; and a purified isoprene composition;further comprising extracting at least one of methanol, acetone, ormethyl acetate from the isoprene-lean solution.